Activation and loading of the starter unit during thiocoraline biosynthesis

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Activation and loading of the starter unit during thiocoraline biosynthesis Shogo Mori, Sanjib K. Shrestha, Javier Fernández, María Álvarez San Millán, Atefeh Garzan, Ahmad H. Al-Mestarihi, Felipe Lombó, and Sylvie Garneau-Tsodikova Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00661 • Publication Date (Web): 01 Aug 2017 Downloaded from http://pubs.acs.org on August 3, 2017

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Biochemistry

Activation and loading of the starter unit during thiocoraline biosynthesis Shogo Mori,a Sanjib K. Shrestha,a Javier Fernández,b María Álvarez San Millán,b Atefeh Garzan,a Ahmad H. Al-Mestarihi,a Felipe Lombó,b and Sylvie Garneau-Tsodikovaa,* a

University of Kentucky, Department of Pharmaceutical Sciences, College of Pharmacy,

Lexington, KY, 40536-0596, USA.

b

Departamento de Biología Funcional e Instituto

Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Oviedo, 33006, Spain. KEYWORDS

Adenylation,

Biosynthesis,

Carrier

protein,

3-Hydroxyquinaldic

acid,

Nonribosomal peptide synthetase.

ACS Paragon Plus Environment

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ABSTRACT

The initiation of the nonribosomal peptide synthetase (NRPS) assembly of the bisintercalator natural product thiocoraline involves key enzymatic steps for the AMP-activation and the carrier protein loading of the starter unit 3-hydroxyquinaldic acid (3HQA). Gene cluster data combined with protein sequence homology analysis originally led us to propose that TioJ could be responsible for the AMP-activation step, whereas TioO could act as the thiolation (T) domain, facilitating the transfer of 3HQA to the next NRPS module, TioR. Herein, we confirmed the involvement of TioJ in thiocoraline biosynthesis by tioJ knockout and in vitro activation of 3HQA studies. However, we demonstrated that the TioJ activated 3HQA does not get loaded onto the T domain TioO, as originally believed, but instead on a fatty acid synthase (FAS) acyl carrier protein (ACP) domain FabC, which is located outside of the thiocoraline gene cluster. We showed a strong interaction between TioJ and FabC. By generating TioJ point mutants mimicking the active site of highly homologous enzymes activating different molecules, we showed that the identity of the substrate activated by adenylation domains such as TioJ is not only determined by the active site residues that directly interact with the substrate. The insights gained from these enzymatic transformations are valuable in the efforts towards deciphering the complete biosynthetic pathway of thiocoraline and bisintercalators in general.


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Biochemistry

INTRODUCTION

Thiocoraline (Figure 1A) is a 2-fold-symmetric bicyclic octathiodepsipeptide bisintercalator secondary metabolite produced by two marine actinomycete strains, Micromonospora sp. ML1 and Micromonospora sp. L-13-ACM2-092.1,

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Among thiocoraline’s distinguishing structural

features are two intercalating 3-hydroxyquinaldic acid (3HQA, 1) moieties (Figures 1A and 2).3 Other bisintercalators structurally related to thiocoraline include BE-22179,4 sandramycin,5 SW163C-G,6 quinaldopeptin,7 and luzopeptins8 also containing 3HQA (1) intercalating units, as well as echinomycin (Figure 1B),9 triostins,10 and quinoxapeptin11 containing quinoxaline-2carboxylic acid (QXC, 2) intercalating moieties (Figures 1B and 2). Thiocoraline displays antimicrobial activity against Gram-positive bacteria and potent antitumor activity against various human cancer cell lines through its DNA bisintercalating properties as well as its DNA polymerase inhibiting activity.12, 13 More recently, thiocoraline was found to activate the Notch pathway in carcinoids and reduce tumor progression in vivo.14 Activation of the Notch pathway has previously been shown to play a role in tumor suppression in slow-growing neuroendocrine tumors.15


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A

TioK/TioT CO2H TioI TioQ NH 2 TioF N H L-Trp

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FabC OH

OH

TioJ

OH N TioL/M O ATP TioG 3-hydroxyquinaldic acid (1) TioH

FabC Sfp, CoA

O

PP i

OH

OAMP

N

S

N O

TioN/TioT TioR/TioT TioS/TioT MeS O O N

B

Ecm13/Ecm8 CO2H Ecm12 Ecm2 NH 2 Ecm11 N H L-Trp

Ecm14 Ecm4 Ecm3

O N

S

N H OH

O N

H N

S O O S

N H

S N

N

O

HO H N

O

N

O O

SMe

Thiocoraline FabC N OH

N O QXC (2)

N

Ecm1

FabC OAMP

N ATP

O

PP i

N Sfp, CoA

S

N O

Ecm6/Ecm8 Ecm7/Ecm8

O O O N N

N O N H

S H N O

O N O S O

N H

N H N O

N

N

N O O

O Echinomycin

Figure 1. A. Representation of the proposed role of TioJ (A domain) in the activation of 3-hydroxyquinaldic acid (3HQA, 1, in dark blue) into its adenylated counterpart followed by its revised loading on FabC ACP domain en route towards thiocoraline formation. B. Representation of the proposed role of Ecm1 (A domain) and FabC (ACP domain) in the biosynthesis of echinomycin using the 3HQA (1) homolog QXC (2). Note: The enzymes utilized in this study are highlighted in blue, turquoise, and gray. The enzymes involved in the formation of cpd 1 and other enzymes involved in thiocoraline production that we previously studied16-20 are highlighted in orange.


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Biochemistry

OH OH

N

OH

N 1 OH

OH

N

O

O

2 N OH

N OH

5

6

O

OH OH

OH

N

O

7

9

OH

10

OH

N

O

8

O

NH O

O

O N H

O

4 Br

H 2N N H

OH

N

O

3

N

O

OH

N

N H 11

OH

O N H 12

OH

OH

O NH O

NH

HN

O O N H 13

OH

N H 14

OH

NH

O HN

O N H 15

OH

N 16

O OH

Figure 2. Chemical structures of the non-amino acid compounds tested against TioJ. Note: The natural substrate of TioJ, 3HQA (1), is in dark blue. Other compounds (2 and 3) that were also found to be substrates of TioJ for which we determined kinetic parameters are in turquoise.

Thiocoraline is assembled mainly by two modules of nonribosomal peptide synthetase (NRPS) enzymes, TioR and TioS (Figure 1A).21 The biosynthesis of nonribosomal peptides typically proceeds through repeating steps based on the catalytic action of three essential components found in each NRPS elongation module: an adenylation (A), a thiolation (T), and a condensation (C) domain. Adenylation (A) domains catalyze the ATP-dependent activation of the carboxylic group of an amino acid (or aryl acid) to form the aminoacyl-AMP intermediate, which is subsequently transferred to the phosphopantetheinyl (Ppant) arm of the active (holo) downstream T domain partner. The amino acid building blocks covalently tethered to successive T domains are then connected by the formation of amide bonds catalyzed by C domains. The two 3HQA (1) planar chromophores appended to the nonribosomal peptide core of thiocoraline have been shown to be derived from L-Trp and believed to be acting as the starter units of the NRPS assembly of this natural product.3 The generation of 1 from L-Trp is proposed to involve eight enzymes (TioK, TioT, TioI, TioQ, TioF, TioL or M, TioG, and TioH) (Figure


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1A and Table S1). The free-standing A-T didomain TioK, co-expressed with its MbtH-like protein partner TioT, was shown to be involved in the ATP-dependent activation and loading of L-Trp

onto its T domain.20 The loaded L-Trp was proposed to undergo β-hydroxylation by the

cytochrome P450 enzyme TioI, and then the β-hydroxylated L-Trp product was demonstrated to be released by the type II thioesterase TioQ.16 The free β-hydroxylated L-Trp in this proposed pathway then undergoes dioxygenation involving the cleavage of its 5-membered pyrrole ring by the Trp 2,3-dioxygenase TioF. In vitro characterization of TioF confirmed its dioxygenase activity in converting L-Trp to N-formylkynurenine and showed that the enzyme is active against other L-Trp analogues such as serotonin, D-Trp, and indole.17 The rest of the enzymatic steps towards the formation of 1 are proposed to involve deformylation of the β-hydroxy-Nformylkynurenine to β-hydroxykynurenine catalyzed by TioL or TioM, followed by cyclization to form a dihydroquinoline ring system by the kynurenine aminotransferase TioG, and finally the oxidoreduction of the quinolone ring ketone by the NADP+-dependent oxidoreductase TioH. Investigations of the remaining uncharacterized enzymatic transformations in this pathway are currently underway in our laboratory. The formation of starter unit 1 triggers the early steps of the NRPS assembly of thiocoraline. We aimed to investigate the incorporation of 1 into the NRPS assembly machinery and decipher the early biosynthetic steps needed to complete this incorporation. To initiate the thiocoraline NRPS assembly, compound 1 must be activated to 1-AMP and then loaded onto an appropriate T domain for easy transfer to the next NRPS module, TioR. Feeding studies in S. triostinicus and S. echinatus showed that QXC (2), a structural analogue of 1, is an intermediate in triostin A and echinomycin biosynthesis (Figure 1B).22,

23

The purified AMP-binding enzyme TrsI from the

triostin biosynthetic pathway was shown to be capable of activating 2 to its 2-AMP counterpart.24


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Biochemistry

In line with this observation, TioJ from the thiocoraline pathway, 62% identity to TrsI, is the likely enzyme to activate 1 to 1-AMP. Unique to the thiocoraline cluster is the presence of the free-standing T domain, TioO, which we originally hypothesized to be the likely candidate for the covalent tethering of 1 prior to its condensation with D-Cys attached to the T1 domain of the next loading module, TioR. In the biosynthesis of echinomycin, triostin A, and SW-163C, it was proposed that an acyl carrier protein (ACP) from the fatty acid biosynthesis enzymatic complex found outside of their gene clusters, FabC, is recruited to play this role.25-27 To confirm these proposed early steps in the NRPS construction of thiocoraline, and to verify if the unique stand-alone T domain TioO (that we and others previously believed to work in concert with the A domain TioJ) or an ACP FabC (as in other bisintercalators’ biosyntheses) is involved in the early stage of thiocoraline biosynthesis, we aimed to heterologously express and purify the enzymes involved in this pathway. Herein, we report the tioJ gene deletion study as well as the in vitro biochemical characterization of the AMP-binding enzyme TioJ. We unambiguously decipher the carrier enzyme used to shuttle the 3HQA (1) starter units during the early stage of thiocoraline production. We also present the interaction of TioJ with its carrier protein partner.

MATERIALS AND METHODS

Bacterial strains, plasmids, materials, and instrumentation. Primers utilized for PCR were bought from Sigma-Aldrich (St. Louis, MO, USA) or from VWR (Radnor, PA, USA). Restriction enzymes, Phusion High-Fidelity DNA polymerase, Pfx DNA polymerase, T4 DNA ligase and all other cloning reagents were purchased from New England Biolabs (NEB, Ipswich, MA, USA) or Invitrogen (Carlsbad, CA, USA). Chemically competent E. coli TOP10 and BL21


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(DE3) cells were purchased from Invitrogen, and BL21-CodonPlus (DE3)-RIL competent cells were purchased from Agilent Technologies (Santa Clara, CA, USA). The E. coli BL21 (DE3)ybdZ::aac(3)IV strain was a generous gift from Professor Michael G. Thomas (University of Wisconsin-Madison, WI, USA). The pET28a and pACYCDuet-1 vectors were purchased from Novagen (EMD Millipore, Billerica, MA, USA). R5A production medium (adjusted to pH 6.85 using KOH) contains per L: sucrose (103 g), K2SO4 (0.25 g), MgCl2•H2O (10.12 g), glucose (10 g), casamino acids (0.1 g), yeast extract (5 g), MOPS (21 g), and trace elements solution (2 mL) (as for R5 medium).28 3-Hydroxyquinoline and 2-methoxyethoxymethyl chloride were purchased from AK Scientific, Inc. (Union City, CA, USA). All other non-radioactive chemicals for synthesis and enzymatic assays were purchased from Sigma-Aldrich or from VWR and used without any further purification. [32P]PPi was purchased from Perkin Elmer (Akron, Ohio, USA). DNA sequencing of all DNA constructs generated was performed at Eurofins MWG Operon LLC (Huntsville, AL, USA). DNA sequencing for the tioJ knockouts was performed at the Sequencing Unit at the SCTs at the Universidad de Oviedo. HPLC analyses monitoring thiocoraline production in S. albus-pFL1049 and S. albus-pFL1049-ΔtioJ were performed using an Agilent 1260 Infinity equipped with a quaternary pump and a 1260 Infinity photo-diode array detector using a C18 Extrasil Teknochroma column (250 × 4.6 mm, 5 µm particle size). HPLC analyses monitoring the loading of 3HQA (1) onto T/ACP domains were performed using a 1200 Infinity Series from Agilent Technologies instrument equipped with a Vydac HPLC DENALI™ column (C18, 250 × 4.6 mm, 5 µm particle size) from Grace (Columbia, MD, USA). Fast protein liquid chromatography (FPLC) was performed for the TioJ/MFabC interaction assay on a BioRad® BioLogic DuoFlow (Bio-Rad®, Hercules, CA, USA) using a HiPrep 26/60 Sephacryl S200 HR column (GE Healthcare, Piscataway, NJ, USA).


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Biochemistry

Generation of a tioJ knockout in S. albus-pFL1049. The preparation of the tioJ mutant strain (ΔtioJ) is depicted in Figure S1. A 1,303-bp DNA fragment in the tioJ gene sequence was PCRamplified using oligonucleotides MAS-J-up (5'-aaggcgacggtcttctcg-3') and MAS-J-rp (5'acaagaccgcactcgtctg-3'). The PCR reaction mixture contained genomic DNA from S. albuspFL1049 (1 µL, 14 ng/µL), the oligonucleotides (20 pmol/µL stock solution) (1.5 µL of each one), dNTPs (2 mM each) (7.5 µL), MgSO4 (50 mM) (1 µL), Pfx reaction buffer (10×) (5 µL), Pfx enhancer solution (10×) (10 µL), distilled H2O (22.5 µL), and Pfx DNA polymerase (0.5 µL). S. albus-pFL1049 is a thiocoraline heterologous producer strain containing the whole 52,908 bp-long biosynthetic gene cluster for thiocoraline integrated by an attP mechanism.21 PCR conditions were: 2 min at 94 °C; 30 cycles (each one including these steps: 30 s at 94 °C, 1 min at 50 °C and 80 s at 68 °C); an elongation step of 5 min at 68 °C; and a final step of 15 min at 4 °C. The PCR product was purified from an agarose gel using a Gel Extraction Kit (QIAGEN), and cloned into pCR-Blunt plasmid vector, giving rise to the pMAS28 plasmid. The pMAS28 plasmid contains the 1,303-bp DNA fragment internal to tioJ (confirmed by sequencing) flanked by EcoRI restriction sites (from pCR-Blunt polylinker). This EcoRI DNA fragment was then subcloned into the pBSKT plasmid vector, generating pMAS36. pBSKT is a pBluescript II SK derivative containing a thiostrepton resistance gene (used as a marker for easy selection of transformant colonies) cloned at the unique NaeI site (outside the polylinker region). pMAS36 was used for transformation of S. albus-pFL1049 protoplasts on R5 Petri dishes, following the classical method,28 and after 24 h the plates were overlayed with 1.5 mL of H2O containing thiostrepton in order to achieve a final concentration of 50 µg thiostrepton/mL of R5. Five


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thiostrepton-resistant colonies were obtained after several transformation experiments with pMAS36. One colony of S. albus-pFL1049-ΔtioJ was inoculated in Bennet medium (with 50 µg thiostrepton/mL) for sporulation, the spores harvested and maintained in a 50% glycerol stock solution at -20 °C, and used for the gDNA and production experiments. One million spores from this tioJ mutant strain were inoculated in a flask containing TSB medium (25 mL), supplemented with 5 µg thiostrepton/mL, 0.75% glycine and MgCl2 (5 mM). After incubation (30 °C, 250 rpm, 72 h) the cells were harvested by centrifugation (8,000 rpm) and used to obtain genomic DNA from this mutant colony using the Salting Out protocol.28 The genomic DNA from S. albuspFL1049, taken as control, and the genomic DNA from S. albus-pFL1049-ΔtioJ were used for testing that tioJ was effectively mutated in this thiostrepton-resistant transformant colony by PCR amplification, using two new oligonucleotides flanking the tioJ gene sequence (JFF-comprΔtioJ-up2: 5’-cgcgtctactcgaacttcct-3’ and JFF-compr-ΔtioJ-rp2: 5’-cgtcctctcggtactgggta-3’). These primers give rise to a 1,893-kb region in wt strain, and to a 7,696-kb region in ΔtioJ strain. PCR conditions in the case of wild-type (wt) strain were: 2 min at 94 °C; 30 cycles (each one including these steps: 30 s at 94 °C, 1 min at 57 °C and 2 min at 68 °C); an elongation step of 5 min at 68 °C; and a final step of 15 min at 4 °C. PCR conditions in the case of ΔtioJ strain were: 2 min at 94 °C; 10 cycles (each one including these steps: 30 s at 94 °C, 1 min at 57 °C and 7 min at 68 °C) plus 15 cycles (each one including these steps: 30 s at 94 °C, 1 min at 57 °C and 9 min at 68 °C); an elongation step of 7 min at 68 °C; and a final step of 15 min at 4 °C. Monitoring of thiocoraline production in S. albus-pFL1049 and S. albus-pFL1049-ΔtioJ. To establish if the S. albus-pFL1049-DtioJ mutant strain can produce thiocoraline, R5A medium (25 mL) supplemented with thiostrepton (5 µg/mL) was inoculated with spores of this mutant


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Biochemistry

strain. After incubation (30 °C, 250 rpm, 4 days), the culture was extracted with two volumes of EtOAc (2×25 mL), and the organic solvent removed in a speed-vac. The dry extract was resuspended in a 1:1/MeOH:DMSO mixture (1 mL) and analyzed by RP-HPLC (injection volume: 10 µL; flow rate: 0.5 mL/min; solvents used: A = H2O (0.1% TFA), B = acetonitrile; gradient used = 10% B for 10 min, 10-99% B over 20 min, 99% B for 10 min, and 10% B for 10 min) (Figure S2A). Product elution was monitored at 230 nm. The retention time for thiocoraline under these conditions was 35.2 min. The production of thiocoraline in the S. albus-pFL1049 control strain was confirmed by ESI-MS in the positive ion mode with a capillary voltage of 3 kV and a cone voltage of 20 V [electrospray ionization for thiocoraline (C48H56N10O12S6): m/z calcd., 1157.4; obs., 1157.3]. No thiocoraline nor thiocoraline intermediates were present in the extract from the S. albus-pFL1049-ΔtioJ mutant strain (Figure S2B). Amino acid alignments of thiocoraline biosynthetic enzymes with other enzymes. Alignments of (i) TioJ with highly homologous A domain enzymes (Figure S3), (ii) T domains TioO from the thiocoraline gene cluster with the ACP domains SlFabC and MFabC (Figure S4), and (iii) SlFabC with FabC homologs from Micromonospora sp. (Figure S5) were generated by MultAlin (http://multalin.toulouse.inra.fr/multalin/).29 Preparation of plasmids for expression and co-expression. For the preparation of Ecm plasmids used in this study, genomic DNA was isolated from Streptomyces lasaliensis ATCC 31180 by a modified cetyltrimethylammonium bromide (CTAB) procedure. Briefly, the bacterial strain was cultured at 26 °C in 5 mL of ISP medium 1 (Fisher Scientific, Hampton, NH, USA) overnight, which was harvested by centrifugation at 3,000 rpm for 15 min at 4 °C. After the supernatant was discarded, the pellet was resuspended in 875 µL of TE25S, which comprises Tris-HCl (25 mM, pH 8.0), EDTA (25 mM), and sucrose (0.3 M), where 17.5 µL of lysozyme


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stock solution (100 mg/mL) was added followed by incubation for 40 min at 37 °C. This was mixed with 8.75 µL of proteinase K stock solution (20 mg/mL), and 50 µL of 10% SDS was subsequently added before incubation for 1 h at 55 °C with occasional inversion. Following the addition of 175 µL of NaCl (4 M), 1.15 µL of CTAB/NaCl mixture (10% CTAB and 0.7 M of NaCl) was added. This thoroughly mixed solution was incubated for 10 min at 55 °C. After cooling it down to 37 °C, 875 µL of phenol:CHCl3:isoamyl alcohol (25:24:1, v/v) was added, and it was incubated for 30 min at 37 °C. The aqueous layer was collected after centrifuge at 10,000 rpm for 15 min where 600 µL of i-PrOH was added. The precipitated DNA was pelleted by centrifugation at 10,000 rpm for 10 min and washed with 250 µL of 70% EtOH. The DNA pellet was air dried and dissolved in 50 µL of TE buffer, which contains Tris-HCl (10 mM, pH 8.0) and EDTA (1 mM). Overexpression constructs of the genes encoding for TioJ and FabC from Micromonospora sp. ML1 (MFabC) were PCR-amplified from genomic DNA of Micromonospora sp. ML1 obtained from PharmaMar (Madrid, Spain), and genes encoding for Ecm1 and FabC from Streptomyces lasaliensis (SlFabC) were PCR-amplified from genomic DNA of Streptomyces lasaliensis ATCC 31180. PCR reactions were performed in mixtures consisting of Phusion High-Fidelity DNA polymerase (0.25 µL), Phusion GC Buffer (5 µL), genomic DNA (15 ng), forward and reverse primers for tioJ and ecm1 (0.25 µL of 50 µM) or for MfabC and SlfabC (0.125 µL of 50 µM), dNTP (0.5 µL of 10 mM), DMSO (1.5 µL), and ddH2O in a total volume of 25 µL. The PCR reaction conditions were optimized as follow: initial denaturation for 30 s at 98 °C, 30 cycles of 30 s at 98 °C, 30 s at 72 °C, and 1 min/1 kb at 72 °C, and a final extension for 10 min at 72 °C. The primers used for the PCR amplification of the genes studied in this work are listed in Table S2 with their corresponding restriction sites underlined. The amplified tioJ and other genes were


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Biochemistry

ligated into the NdeI/XhoI and NdeI/HindIII restriction sites, respectively, of the linearized pET28a to give the constructs pTioJ-pET28a, pMFabC-pET28a, pEcm1-pET28a, and pSlFabCpET28a encoding NHis6-tagged proteins. The ligation products were transformed into E. coli TOP10 chemically competent cells, which were grown on a Luria-Bertani (LB) medium plate supplemented with kanamycin (50 µg/mL). These constructs were confirmed by DNA sequencing. Based on the amino acid residues in the active sites of TioJ and its homologs involved in dictating the identity of the substrate to be activated, we decided to generate the single point mutants TioJN235Q and TioJN235D to evaluate their substrate specificity. The primers utilized to generate the single point mutants and the cloning strategies are presented in Tables S1 and S2, respectively. The pTioJ-pET28a plasmid was used as a template to amplify DNA fragments tioJN235Q overhang fwd (fragment a, as indicated in Table S3) and tioJN235Q overhang rev (fragment b) as well as DNA fragments tioJN235D overhang fwd (fragment d) and tioJN235D overhang rev (fragment e) with one round of PCR for each fragment by using the PCR primers listed in Table S2. In the second round of PCR, gel purified PCR products fragment a (~0.71 kb) and fragment b (~0.9 kb) as well as fragment d (~0.71 kb) and fragment e (~0.9 kb) were used as templates to produce full-length tioJN235Q (fragment c, ~1.6 kb) and tioJN235D (fragment f, ~1.6 kb), respectively, by using the PCR primers listed in Table S2. The amplified fragments c and f (~1.6 kb) were then double digested and subcloned into the linearized pET28a vector via the corresponding NdeI/XhoI restriction sites to produce the final overexpression constructs pTioJN235Q-pET28a and pTioJN235D-pET28a vectors encoding NHis6-tagged proteins. All cloning experiments were done in E. coli TOP10 cells. The expression clone was confirmed by DNA sequencing.


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Overexpression and purification of TioJ, TioJ co-expressed with TioT, TioJ mutants (N235Q and N235D), Ecm1, TioO, SlFabC, and MFabC. The TioO (T domain) protein was purified as previously described.16 For overexpression of each protein, pTioJ-pET28a, pTioJN235Q-pET28a, pTioJN235D-pET28a, pEcm1-pET28a, and pSlFabC-pET28a were transformed into chemically competent BL21 (DE3), and pMFabC-pET28a was transformed into chemically competent BL21 (DE3)RIL. For co-expression of TioJ with its MbtH-like protein partner TioT (for a control experiment to verify if TioT is needed for expression and/or activity of TioJ), the cloned construct pTioJ-pET28a was transformed into chemically competent BL21 (DE3)ybdZ::aac(3)IV with pTioT-pACYCDuet-1.20 The transformants were incubated overnight at 37 °C in 3×5 mL of LB medium supplemented with kanamycin (50 µg/mL) for overexpression of TioJ, TioJ mutants (N235Q and N235D), Ecm1, TioO, and SlFabC or with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL) for overexpression of TioJ/TioT and MFabC. Each overnight culture was inoculated in LB medium (3×1 L) supplemented with MgCl2 (10 mM) for overexpression of TioJ, TioJ mutants (N235Q and N235D), TioJ/TioT, and Ecm1 and with corresponding antibiotics until it reached an OD600 of 0.5-0.7. The culture was then cooled to 16 °C before induction with isopropryl-β-D-1-thiogalactopyranoside (IPTG, 0.2 mM) and shaken for an additional 20 h at 16 °C. Cells were harvested by centrifugation at 5,000 rpm for 10 min at 4 °C. After washing the cells with ddH2O and buffer A, which contains Tris-HCl (25 mM, pH 8.0 adjusted at rt), NaCl (400 mM), imidazole (5 mM), and glycerol (10% v/v), it was resuspended in 40 mL of buffer A supplemented with phenylmethylsulfonyl fluoride (PMSF, 1 mM) and dithiothreitol (DTT, 1 mM). The resuspended cells were lysed by 4 cycles of sonication (130 s with 10 s ‘on’ alternating with 20 s ‘off’) and the cell debris was removed by centrifugation at 16,000 rpm for 45 min at 4 °C. The protein solution was incubated with 0.75


14

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Biochemistry

mL of NiII-NTA agarose resin (Qiagen, Gaithersburg, MD, USA) at 4 °C for 2 h with gentle rocking. The resin was loaded onto a column and washed with 10×10 mL of buffer A supplemented with additional imidazole (40 mM final). The proteins were eluted from the column with 3×5 mL of buffer A supplemented with additional imidazole (500 mM final). For all the proteins, with the exception of MFabC, the first two elution fractions were combined and dialyzed three times (at least 3 h for each dialysis, total 20 h) at 4 °C against 2 L of buffer B, which contains Tris-HCl (40 mM, pH 8.0 adjusted at rt), NaCl (200 mM), β-mercaptoethanol (2 mM) and glycerol (10% v/v) using SnakeSkin Dialysis Tubing, 10K MWCO for TioJ, TioJ mutants, and Ecm1, as well as 3K MWCO for TioJ/TioT, TioO, and SlFabC (Thermo Scientific). In the case of MFabC, the first elution fraction was injected onto a HiPrep 26/60 Sephacryl S200 HR FPLC column using buffer B without glycerol. The fractions containing pure MFabC, as determined by SDS-PAGE analysis, were combined, and glycerol (10% v/v) was added. The dialyzed or FPLC purified proteins were concentrated using Amicon Ultra-15 Centrifugal Filter Units (EMD Millipore, Billerica, MA, USA) with 10K MWCO membrane for TioJ, TioJ mutants, and Ecm1, or 3K MWCO membrane for TioJ/TioT, TioO, SlFabC, and MFabC, flashfrozen in liquid nitrogen, and stored at -80 °C. The yields of each protein were as follow: TioJ: 4.1 mg/L of culture, TioJ/TioT: 2.8 mg/L of culture, TioJN235Q: 0.24 mg/L of culture, TioJN235D: 0.53 mg/L of culture, Ecm1: 4.9 mg/L of culture, TioO: 4.2 mg/L of culture, SlFabC: 0.08 mg/L of culture, and MFabC: 0.18 mg/L of culture. Determination of the substrate specificity and kinetic parameters of adenylation activity using ATP-[32P]PPi exchange assays. To establish the substrate specificity profile for TioJ (Figure 3A and 3Bb), Ecm1 (Figure 3Ba), TioJN235Q (Figure 3Bc), and TioJN235D (Figure 3Bd), ATP-[32P]PPi exchange assays were performed for 2 h at rt. Briefly, reactions (100 µL)


15

Biochemistry

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Page 16 of 45

containing Tris-HCl (75 mM, pH 7.5 adjusted at rt), TCEP (1 mM, pH 7.0), MgCl2 (10 mM), ATP (5 mM), Na4P2O7 (1 mM, containing about 500,000 cpm of [32P]PPi per reaction), substrate (1 mM), and DMSO (5% final concentration) were initiated by addition of enzyme (2.5 µM). After 2 h of incubation at rt, reactions were quenched with 500 µL of quenching solution (1.6% (w/v) activated charcoal, 4.5% (w/v) Na4P2O7, and 3.5% (v/v) perchloric acid in H2O). The charcoal was pelleted by centrifugation at 14,000 rpm for 7 min at rt, and then washed with wash solution (4.5% (w/v) Na4P2O7, and 3.5% (v/v) perchloric acid in H2O). The pellet was then resuspended in 500 µL of H2O followed by resuspension in 5 mL of scintillation cocktail, whose radioactivity was measured by a liquid scintillation counter. For the determination of the steady-state kinetic parameters of adenylation (Figure S6 and Table 2), the ATP-[32P]PPi exchange assay described above was performed with varying substrate concentrations. Reactions (100 µL for each individual substrate concentration) were prepared in the same manner as described above for substrate profiling, except for enzyme concentrations (1 µM of TioJ, 5 µM of TioJN235Q, 2.5 µM of TioJN235D, and 0.25 µM of Ecm1). All reactions were stopped after 12 min for TioJ with all three substrates (3HQA (1), QXC (2), and QA (3)), 11 min for Ecm1 with QXC (2) and QA (3), 60 min for Ecm1 with KA (4), 12 min for TioJN235Q with 3HQA (1) and QXC (2), and 45 min for TioJN235D with 3HQA (1). Processing and counting of [32P]ATP were performed as described above in this section. Characterization of SlFabC and MFabC activity by trichloroacetic acid (TCA) precipitation assays. To characterize the activity of SlFabC and MFabC, conversion of the protein from its apo (inactive) to holo (active) form was monitored in presence of Sfp (phosphopantetheinyltransferase) and [3H]AcCoA. Incorporation of [3H]acetyl into the apo


16

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Biochemistry

proteins over time was determined by using TCA precipitation assays at rt as previously described.19 The total 25-µL reaction containing Tris-HCl (75 mM, pH 7.5 adjusted at rt), MgCl2 (10 mM), TCEP (1 mM), AcCoA (100 µM, spiked with ~150,000 cpm of [3H]AcCoA), apo SlFabC or MFabC (15 µM) was initiated by addition of Sfp (1 µM). The reaction was quenched by adding 100 µL of a 10% TCA solution at 0, 2, 5, 10, 30, and 60 min. The precipitate was pelleted by centrifugation at 14,000 rpm for 7 min at rt, and washed twice with 100 µL of a 10% TCA solution. After the final wash, the pellets were dissolved into 100 µL of 88% formic acid. This was transferred into liquid scintillation vials containing 5 mL of the scintillation fluid, and the radiation activity was measured by liquid scintillation counter (Figure S7). HPLC analysis of loading of 3-hydroxyquinaldic acid (3HQA, 1) activated by TioJ onto T/ACP domains. To monitor the loading of 3HQA (1) onto free-standing T/ACP domains (TioO, SlFabC, and MFabC), two reaction mixtures were prepared. The first reaction mixture was to activate 3HQA (1) by TioJ where the 50-µL reaction solution containing Tris-HCl (75 mM, pH 7.5 adjusted at rt), MgCl2 (10 mM), TCEP (1 mM), ATP (1 mM), 3HQA (1, 1 mM), DMSO (5% v/v), TioJ (5 µM) was incubated for 2 h at rt. Meanwhile, the other 50-µL reaction mixture containing Tris-HCl (75 mM, pH 7.5), MgCl2 (10 mM), TCEP (1 mM), CoA (0.5 mM), Sfp (2.5 µM), T/ACP domain (100 µM) to produce a holo T/ACP domain was incubated for 30 min at rt. The loading reaction was initiated by mixing the above two reaction mixtures and incubated at rt for 0, 0.5, 1, 2, 5, 15, 30, and 60 min for the time course assay or for 30 min for the T/ACP domain profile assay. The reaction was quenched by adding 400 µL of ice-cold MeOH and incubated for 2 h at -20 °C. The precipitated proteins were pelleted by centrifugation at 13,000 rpm for 10 min at 4 °C and washed with 500 µL of 80% ice-cold MeOH three times. The washed pellet was air dried, and 100 µL of 0.1 N KOH was added to completely dissolve the


17

Biochemistry

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pellet. This was heated for 30 min at 60 °C and acidified with 2 µL of 6 N HCl. 3HQA (1) was extracted with 4×100 µL of EtOAc which was evaporated off by a gentle flow of N2. The dried 3HQA (1) was dissolved in 20 µL of H2O, and 15 µL of this was injected onto HPLC. HPLC was run by using the following method; flow rate = 1 mL/min, solvent A = H2O with 0.1% TFA, solvent B = MeCN, gradient = 0% B for 5 min, increase to 20% B over the next 15 min (time 520 min on the chromatogram), increase to 60% B over the next 5 min (time 20-25 min on the chromatogram), increase to 100% B over the next 1 min (time 25-26 min on the chromatogram), stay at 100% B for 5 min (time 26-31 min on the chromatogram), decrease to 0% B in 1 min and stay at 0% B for 8 min. The 3HQA (1) peak was monitored at a wavelength of 254 nm and found at a Rt of 23.7 min (Figures 4 and 5). Evaluation of the interaction between TioJ and MFabC. To assess the interaction between TioJ and MFabC, FPLC fractions containing TioJ, MFabC, or TioJ/MFabC mixture were collected and analyzed by SDS-PAGE. TioJ and MFabC were expressed and purified as described above. Instead of dialyzing the proteins, they (5.5 mL) were loaded onto a HiPrep 26/60 Sephacryl S-200 HR FPLC column using a buffer containing Tris-HCl (40 mM, pH 8.0 adjusted at rt), NaCl (100 mM), and b-mercaptoethanol (2 mM). When loading individually, FPLC fractions identified to contain TioJ (fractions 13 and 14, indicated by (i) in Figure S8A) and MFabC (fractions 20-22, indicated by (ii) in Figure S8B) by monitoring at a wavelength of 280 nm, were collected and analyzed by SDS-PAGE (Figure S8D). To confirm that aggregation of MFabC was not present in the fractions 13 and 14 corresponding to TioJ, we collected fractions 13 and 14 from gel filtration of MFabC (indicated by (iii) in Figure S8B) and concentrated them to 0.5 mL. Doing so, we confirmed that there was no MFabC aggregation. The fractions for each protein were concentrated to 3 mL using an Amicon Ultra-15 Centrifugal


18

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Biochemistry

Filter Unit (EMD Millipore, Billerica, MA, USA) with 10 K and 3 K MWCO for TioJ and MFabC, respectively. The concentrated proteins were mixed together in a 1:1 ratio and incubated for 1 h on ice prior to be loaded onto the FPLC column again. Fractions 13 and 14 (indicated by (iv) in Figure S8C) were collected, concentrated to 0.5 mL, and analyzed by SDS-PAGE (Figure S8D), and interaction between TioJ and MFabC was observed. Chemical synthesis and characterization of compounds to be used as potential TioJ substrates. The 3-hydroxyquinaldic acid (1) was generated following the scheme presented in Figure S9. Synthesis of compound 17. Sodium hydride (0.36 g, 8.98 mmol) was slowly added to a solution of 3-hydroxyquinoline (1.00 g, 6.89 mmol) in THF (120 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min after which methoxyethoxymethyl chloride (1.27 mL, 11.04 mmol) was added. The reaction mixture was slowly warmed to rt and stirred for 12 h. The mixture was diluted with EtOAc, washed with brine, dried over MgSO4, and concentrated under reduced

pressure.

The

residue

was

purified

by

column

chromatography

(SiO2,

MeOH:CH2Cl2/5:95, Rf 0.21), to give compound 17 (1.02 g, 63%) as a red liquid: 1H NMR (400 MHz, (CDCl3) δ 8.69 (br s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.71 (s, 2H), 7.54 (t, J = 7.6 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 5.37 (s, 2H), 3.84 (t, J = 3.6 Hz, 2H), 3.53 (t, J = 3.6 Hz, 2H), 3.33 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 150.4, 144.5, 143.9, 128.9, 128.6, 127.0, 126.9, 116.5, 93.6, 71.3, 67.8, 58.9; LRMS m/z calcd for C13H16NO3 [M+H]+: 234.1; found 234.8. Synthesis of compound 18. The new compound 18 was prepared following a previously published protocol for a similar compound.30 Compound 17 (0.50 g, 2.15 mmol) was dissolved in dry THF (6 mL) and methyllithium (1.6 M in Et2O, 1.57 mL, 2.51 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 1 h, until all the starting material disappeared. The


19

Biochemistry

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reaction mixture was treated with a saturated aqueous solution of NH4Cl (5 mL) and extracted with CH2Cl2 (3×15 mL). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in acetone (1 mL) and treated with an aqueous solution of ammonium cerium (IV) nitrate (2.32 g, 4.24 mmol, in 6 mL) for 30 min. The mixture was extracted with CH2Cl2 (3×15 mL) and dried over MgSO4, and the filtrate was concentrated under reduced pressure to afford 18 (0.48 g, 91%) as an orange solid that was used without any further purification: 1H NMR (400 MHz, (CDCl3) δ 8.50 (d, J = 7.6 Hz, 1H), 8.33 (s, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 5.54 (s, 2H), 3.89 (br t, J = 4.0 Hz, 2H), 3.56 (br t, J = 4.0 Hz, 2H), 3.34 (s, 3H), 3.00 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 152.0, 149.6, 134.3, 131.6, 129.6, 128.0, 127.2, 124.1, 121.5, 94.4, 71.3, 68.9, 59.0, 16.2; LRMS m/z calcd for C14H18NO3 [M+H]+: 248.1; found 248.8. Synthesis of compound 1. The known compound 2 (3-hydroxyquinaldic acid) was prepared following a previously published protocol.30 Compound 18 (0.20 g, 0.81 mmol) was dissolved in dry 1,4-dioxane (8 mL) and selenium dioxide (0.10 g, 0.86 mmol) was added. The reaction mixture was heated under reflux for 1 h and then cooled and filtered through Celite®. The solution was concentrated under reduced pressure. The residue was dissolved in formic acid (0.3 mL) and treated with hydrogen peroxide (30% solution, 0.33 g, 4.05 mmol) at 0 °C for 12 h. The mixture was filtered and washed with cold H2O to afford the pure compound 1 (0.02 g, 13%) as a yellow solid: 1H NMR (400 MHz, (CD3)2SO, which matches lit.30) δ 8.14 (d, J = 8.0 Hz, 1H), 8.02 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.71-7.64 (m, 2H).

RESULTS AND DISCUSSION


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Biochemistry

Sequence homology of TioJ with other aryl substrate-activating A domains. In addition to its 62% identity to TrsI, the amino acid sequence of TioJ is homologous to other aryl substrateactivating A domains such as Qui16 (62% identity) from the echinomycin pathway in Streptomyces griseovariabilis, Ecm1 (65% identity) from the echinomycin pathway in Streptomyces lasaliensis, and Swb12 (61% identity) from the SW-163D pathway in Streptomyces sp. SNA15896. Interestingly, inspection of the substrate specificity determining residues, based on the A domain code,31 revealed that only the amino acid residue at position 235 appears to play a role in differentiation between substrates 1 and 2 (Table 1). In TioJ and Swb12, known to activate compound 1, an asparagine (N) is found at position 235, whereas a glutamine (Q) is found at this position in TrsI, Qui16, and Ecm1, known to activate compound 2. However, as the rest of the A domain code for all of these enzymes is basically identical, and as compounds 1 and 2 are very similar in structures, we believed that both of these compounds are possible substrates of TioJ, which we explored in this study (see substrate specificity profile of TioJ section below). Table 1. Substrate specificity determining residues in the active site adenylate-binding pocket of A domains involved in activation of compounds 1 and 2. Enzyme Substrate Amino acid positiona 235 236 239 278 299 301 322 330 331 517 TioJ 1 N S C T Q G W L T K Swb12 1 N A C T Q G W L T K TrsIb 2 Q A C T Q G W L T K Qui16 2 Q A C T Q G W L T K Ecm1 2 Q A C T Q G W L T K a Residue numbers used are based on the crystal structure of the gramicidin S synthase 1 A domain previously reported.31, 32 b The code for TrsI was previously reported and was used for alignment of amino acid residues in this study.33

Dependence of thiocoraline production on TioJ. To test the involvement of TioJ in thiocoraline production, we first prepared a tioJ knockout in the thiocoraline-overproducing strain Streptomyces albus-pFL1049, S. albus-pFL1049-ΔtioJ (Figure S1). Using HPLC and mass spectrometry, we confirmed that this ΔtioJ strain does not produce thiocoraline (Figure S2),


21

Biochemistry

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providing a proof that TioJ is directly involved in the biosynthesis of this bisintercalator. Additionally, no thiocoraline intermediates were detected in the S. albus strain lacking the tioJ gene, supporting the proposed role of TioJ in the activation of the starter unit 1, which triggers the complete NRPS assembly of thiocoraline. Heterologous expression of TioJ, Ecm1, TioO, and FabC enzymes. The activation and the subsequent loading steps of the starter unit 1 in thiocoraline biosynthesis are anticipated to require the participation of TioJ for the AMP-activation step and either TioO (unique to the thiocoraline gene cluster) or FabC (present in many bisintercalator producing organisms), which acts as the carrier protein facilitating the subsequent condensation of 1 with the next amino acid building block,

D-Cys,

on the next NRPS module TioR. As we and others previously

hypothesized that TioO would play the carrier role, we first expressed and purified this T domain as we previously described.16 We cloned the PCR amplified tioJ gene into a pET28a vector for heterologous expression and purification in E. coli BL21 (DE3). Our initial attempts (varying IPTG induction concentration, temperature, and incubation period) were unsuccessful. Some NRPS A domains are known to require an MbtH-like partner for solubility, activity, or both.34-36 We previously found that the MbtH-like protein KtzJ is required for soluble protein production and activity of the interrupted A4 domain of KtzH during kutzneride biosynthesis.37 Similarly, we showed that the MbtH-like protein TioT is required for the production of active TioN and the production of soluble and active TioK, other A domains involved in thiocoraline biosynthesis.19, 20 We therefore thought that it is possible that TioJ would require TioT as its MbtH-like partner for optimal expression and activity. However, we found that co-expression with TioT did not allow for soluble production of TioJ. Next, by comparing the proposed amino acid sequence of TioJ that we had


22

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Biochemistry

cloned with those of other homologous enzymes, Swb12, TrsI, Qui16, and Ecm1, we observed that TioJ might have five amino acids missing at its N-terminus for soluble expression (Figure S3). The nucleotide sequence, which would express the identical amino acid sequence to homologous enzymes, was found in front of the proposed tioJ gene in the genomic DNA. By constructing pTioJ-pET28a with these extra five amino acid residues, we were able to isolate TioJ in its soluble and active form. When we co-expressed this longer TioJ construct with TioT, we observed no differences in production or activity of the enzyme. For this study, we also cloned Ecm1 and two FabC enzymes (FabC cloned from Streptomyces lasaliensis: SlFabC, and from Micromonospora sp. ML1: MFabC) into pET28a. The Ecm1 and SlFabC enzymes were overexpressed in E. coli BL21 (DE3), whereas MFabC was overexpressed in BL21-CodonPlus (DE3)-RIL. Substrate specificity and kinetic parameters of TioJ. To investigate the substrate profile of the adenylating enzyme TioJ, we used the well-established ATP-[32P]PPi exchange assay, which allows for the indirect monitoring of the formation of the substrate-AMP product through the reversible exchange of [32P]PPi with ATP. Commercially available and chemically synthesized compounds tested in this assay are shown in Figure 2. As predicted, compound 1 displayed the best activity among all substrates tested (Figure 3A), and compounds 2 and 3, as proposed based on the A domain code of TioJ, were also found to be good substrates of this enzyme. Interestingly compounds 4-6, which are also structurally related to 1 and 2, were found not to be good substrates of TioJ, indicating that this enzyme does not accept compounds with hydroxyl at their 4-position or a combination of nitrogen and hydroxyl at the 4- and 3-positions, respectively. All pyrrole acid analogues and common amino acids tested were also found to be poor substrates of TioJ.


23

Biochemistry

A 100

Relative % activity

80 60 40

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 L-Ala L-Arg L-Asn L-Asp L-Cys L-Gln L-Glu L-Gly L-His L-Ile L-Leu L-Lys L-Met L-Phe L-Pro L-Ser L-Thr L-Try L-Tyr L-Val

20

Substrate

B

a

b

c

d

100 Relative % activity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 45

80 60 40 20 0

1 2 3 4 5 6

1

1 2 3 4 5 6 Substrate

1 2 3 4 5 6

Figure 3. Relative substrate specificity of A. TioJ, Ba. Ecm1, Bb. TioJ, Bc. TioJN235Q, and Bd. TioJN235D as determined by ATP-[32P]PPi exchange assays. Relative activity of TioJ mutants was calculated as the natural substrate (1) of TioJ (Bb) is 100%. The data were obtained in 2-h end-point assays.

Next, we used the same assay to derive the Michaelis-Menten steady-state kinetic parameters (Km and kcat) of adenylation for 1-3 for which TioJ displayed activity during the determination of its substrate profile (Table 2 and Figure S6). TioJ displayed very high catalytic efficiency with 1 (kcat/Km = 10,677 ± 1,549 mM-1min-1) when compared to that of other amino acid-activating A domains in thiocoraline biosynthesis, such as TioN (kcat/Km = 44 ± 6 mM-1min-1)19 and TioK (kcat/Km = 49 ± 8 mM-1min-1)20 against their respective natural substrates. The higher catalytic efficiency of TioJ with 1 results from the substrate affinity of the enzyme for this compound (Km = 0.21 ± 0.03 µM), which is much better than that observed for other A domains with their


24

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Biochemistry

natural substrates (e.g., Km = 45 ± 6 µM and 95 ± 16 µM for TioN and TioK, respectively). This was also much superior to its affinity for 2 (Km = 146 ± 11 µM) which showed 98% activity in the substrate profile. This resulted in a 380-fold decrease in catalytic efficiency for 2 (kcat/Km = 28 ± 2) when compared to that of 1. When looking at the kinetic parameters for compound 3, we found it to be a worse substrate of TioJ than 1 (43-fold lower kcat/Km), but better than 2 (9-fold higher kcat/Km), as a result of varied Km values. It is to note that the turnover rate (kcat) of 1, 2, and 3 were highly similar. This kinetic parameter study confirmed that compound 1 is the natural substrate for TioJ. Table 2. Steady-state kinetic parameters for AMP derivatization by TioJ. Protein Substrate Km (µM) TioJ 3HQA (1) 0.21 ± 0.03 QXC (2) 147 ± 11 QA (3) 5.6 ± 0.4 Ecm1 QXC (2) 5.0 ± 0.1 QA (3) 101 ± 6 KA (4) 1,824 ± 100 TioJ N235Q 3HQA (1) 2.4 ± 0.4 QXC (2) 217 ± 74 TioJ N235D 3HQA (1) 31 ± 4

kcat (min-1) 2.2 ± 0.1 4.2 ± 0.1 1.40 ± 0.02 16.1 ± 0.1 29.7 ± 0.5 0.37 ± 0.01 0.28 ± 0.02 0.32 ± 0.04 0.42 ± 0.01

kcat/Km (mM-1min-1) 10,677 ± 1,549 28 ± 2 249 ± 20 3,205 ± 51 295 ± 18 0.20 ± 0.01 120 ± 20 1.5 ± 0.5 14 ± 2

Mutational study of TioJ and comparison with Ecm1. By analysis of the A domain code for TioJ and other related A domains (Table 1), we postulated that the amino acid residue at position 235 could be responsible for directing the substrate specificity of TioJ for compound 1 over that of compound 2. By crystallography, an asparagine at position 235 (N235) of DhbE, an A domain known to activate 2,3-dihydroxybenzoic acid, was previously reported to play a critical role in binding to the 2-hydroxyl group of this substrate.38 To scrutinize a possibility of substrate swapping from compound 1 to 2, TioJ mutants were produced to mimic the active site of Ecm1, known to activate compound 2. The N235 residue of TioJ was mutated to Q, which is the amino acid residue at position 235 in Ecm1, as well as to D, which is an amino acid seen in many A


25

Biochemistry

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domains whose substrates are carboxylic acids. By substrate profile and kinetic studies, Ecm1 was first confirmed to favor QXC (2) as its natural substrate over compounds 3 and 4, and to not accept 3HQA (1) as a substrate (Figure 3Ba and Table 2). In contrary to what we predicted, the TioJN235Q and TioJN235D mutants did not activate QXC (2), but instead only lowered the activity of TioJ towards their natural substrate 3HQA (1) (Figure 3Bb-d and Table 2). These results indicate that only mimicking the active site residues of an A domain is not sufficient to confer a change in its substrate specificity, even when the rest of the predicted specificity-dictating amino acid residues are basically identical. In addition to the substrate recognition site, the structural conformation of the enzyme may play a crucial role in dictating specificity. A similar phenomenon was observed in a study that produced a new nonnatural “natural” product by site-directed mutagenesis on a surface residue of an A domain.39 Loading of the activated 3-HQA (1) onto an ACP domain of fatty acid synthase, FabC. Having established that 3HQA (1) is indeed the natural substrate of TioJ, we next explored its loading onto a T domain. Since there is only one free-standing T domain, TioO, in the thiocoraline biosynthetic gene cluster, we postulated that it would be the T domain partner of TioJ used to initiate the assembly of thiocoraline.21 T domains are usually expressed in their inactive (apo) form, and need to be posttranslationally modified to their active (holo) form by a phosphopantetheinyltransferase (PPTase) enzyme. PPTases transfer the Ppant moiety of CoA onto an active site serine residue of T domains. We previously showed the apo to holo conversion of TioO by using a common promiscuous PPTase, Sfp.16 Despite many efforts, we never could covalently tether the activated 3HQA (1-AMP) onto holo TioO (Figure 4).


26

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Biochemistry

3HQA (1) std MFabC SlFabC TioO No T domain 21

22

23

24 25 Time (min)

26

27

28

Figure 4. RP-HPLC analysis of loading of 3HQA (1) onto different T domains.

As a result, we hypothesized that TioO was in fact not the T domain partner of TioJ, but that there could be another carrier protein (e.g., MFabC) in the thiocoraline-producing organism that could embark 3HQA (1) into the biosynthetic assembly-line for the production of this bisintercalator. This hypothesis was bioinformatically supported by the fact that MFabC found outside of the thiocoraline gene cluster in the genomic DNA of Micromonospora sp. is highly homologous in amino acid sequence to FabC from S. lasaliensis and other Micromonospora (Figure S5). FabC is known to be involved in other bisintercalators’ biosyntheses. To explore this hypothesis, we cloned and overproduced FabC from the thiocoraline producing strain, Micromonospora sp. ML1 (MFabC), as well as FabC from S. lasaliensis (SlFabC), which is the echinomycin producing strain. These two ACP domains were shown to be converted to their holo (active) form by Sfp using [3H]acetyl-CoA as a conversion marker (Figure S7). Using holo MFabC and SlFabC, we showed by RP-HPLC that the activated 3HQA (1-AMP) can be


27

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 45

covalently attached to both of these ACPs, confirming that MFabC, and not TioO, is the carrier protein involved in thiocoraline biosynthesis (Figure 4). By performing a time-course experiment monitoring the loading of 3QHA (1) onto MFabC, we demonstrated the gradual successful loading of 1 onto MFabC overtime, as expected, which reached completion within 30 minutes (Figure 5). These HPLC experiments demonstrated that the interactions between TioJ and carrier proteins are specific enough to distinguish fatty acid synthase (FAS) ACP domains (e.g., MFabC and SlFabC) from NRPS T domains (e.g., TioO). Taken together, these data indicate that the pathway of activation and loading of 1 proceeds first via its TioJ-catalyzed ATP-dependent adenylation into 1-AMP prior to loading of this activated substrate onto the FAS ACS domain located outside of the thiocoraline biosynthetic gene cluster, MFabC.


28

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

3HQA (1) std 60 min 30 min 15 min 5 min 2 min 1 min 0.5 min 0 min 21

22

23

24 25 Time (min)

26

27

28

Figure 5. RP-HPLC analysis of loading of 3HQA (1) onto MFabC in time course.

Interaction between TioJ and MFabC. To confirm the interaction between the stand-alone TioJ and MFabC enzymes, we performed FPLC and SDS-PAGE analyses (Figure S8). Purified TioJ and MFabC were first individually injected onto an FPLC column to establish that these two enzymes did not elute in the same FPLC fractions. TioJ eluted in fractions 13 and 14 (indicated by (i) in Figure S8A), whereas MFabC eluted in fractions 20-22 (indicated by (ii) in Figure S8B). The FPLC purified proteins were then mixed together and incubated for one hour on ice prior to another injection onto an FPLC column. The small 10.6-kDa MFabC and the large 59.6-kDa TioJ were found to elute as a complex in fractions 13 and 14 (indicated by (iv) in Figure S8C),


29

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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further confirming a strong interaction between these two enzymes. These data provided additional support for MFabC being the carrier protein partner of TioJ.

CONCLUSION

In summary, this study provided very useful insights into the catalysis of the initiating steps of the NRPS assembly enzymes TioJ and the carrier protein MFabC in the biosynthesis of thiocoraline. In this work, we presented biochemical validation of key steps in the biosynthetic pathway of thiocoraline and determined the substrate specificities of the AMP-activating A domain, TioJ, whose functional amino acid sequence was revised. The next step of the assemblyline, loading of 3HQA (1) onto a carrier protein, was also revised as the partner of TioJ was discovered to be MFabC instead of the proposed stand-alone T domain, TioO, which was originally proposed. TioJ and MFabC showed strong interaction as an A-ACP complex. TioJ mutants, which mimic the active site of its highly homologous enzymes Ecm1, Qui16, and TrsI known to activate QXC (2), showed that the substrate to be activated by the A domain is not selected only based on the ten proposed residues that would directly interact with the substrate, but also by the overall structure of the enzyme. The knowledge gained from understanding the catalysis of these enzymes is a major contribution towards the creation of a vast array of novel thiocoraline analogues, some of which could possess improved pharmacological properties for therapeutic applications. Future work will delve into studying the interplay between these enzymes and the downstream NRPS assembly modules in the thiocoraline pathway to provide a better understanding of the molecular requirements of thiocoraline’s NRPS assembly.

ASSOCIATED CONTENT


30

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Biochemistry

Supporting Information. Supporting Tables and Figures are presented in the supporting information. A table of enzymes involved in the production, activation, and loading of 3HQA (Table S1), as well as tables of primers used for the amplification of the tioJ genes (Table S2) and tioJ mutants (Table S3) that we cloned are provided. Figures showing the generation of the tioJ mutant strain (Figure S1), the HPLC traces monitoring thiocoraline production (Figure S2), alignment of TioJ with highly homologous enzymes (Figure S3), alignment of TioO, SlFabC, and MFabC (Figure S4), alignment of SlFabC and its homologous enzymes in Micromonospora sp. (Figure S5), the Michaelis-Menten kinetic parameters of the TioJ, Ecm1, TioJ mutantscatalyzed adenylation of compounds 1-4 (Figure S6), Conversion of inactive apo SlFabC and MFabC to the active holo forms (Figure S7), FPLC and SDS-PAGE analysis for TioJ/MFabC interaction (Figure S8), and the synthetic scheme for the preparation of compound 1 (Figure S9) are also provided. The following files are available free of charge (PDF). AUTHOR INFORMATION Corresponding Author * [email protected] Author Contributions S.M. and S.G.-T. designed the study and wrote the manuscript. S.M. did all of the work, with the exception of that listed next. S.K.S. cloned and characterized the TioJ mutants. J.F., M.A.S.M., and F.L. performed the TioJ gene deletion experiments. A.G. synthesized the compounds tested in the substrate profile of TioJ, TioJ mutants, and Ecm1. A.H.A.-M. performed preliminary experiments with TioJ and TioO and helped with some of the writing. The manuscript was


31

Biochemistry

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Page 32 of 45

written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This project was supported by a NSF CAREER Award MCB-1149427 (to S.G.-T.), a grant PEST08-17 from the Fundación para la Investigación Científica y Tecnológica, Government of the Principality of Asturias, Spain (to F.L.), and startup funds from the College of Pharmacy at the University of Kentucky (to S.G.-T.). J.F. was supported by a fellowship (14-RTC-20141525-2) from the Ministerio de Economía y Competitividad, Government of Spain. ACKNOWLEDGMENT This work was supported by an NSF CAREER Award MCB-1149427 (to S.G.-T.), a grant PEST08-17 from the Fundación para la Investigación Científica y Tecnológica, Government of the Principality of Asturias, Spain (to F.L.), and startup funds from the College of Pharmacy at the University of Kentucky (to S.G.-T.). J.F. was supported by a fellowship (14-RTC-20141525-2) from the Ministerio de Economía y Competitividad, Government of Spain. We thank Taylor A. Lundy for isolating genomic DNA of Streptomyces lasaliensis, Olga E. Zolova for participating in the early stage of cloning of some TioJ constructs, and Josephine M. Kim for participating in the early stage of biochemical assays. ABBREVIATIONS ACP, acyl carrier protein; AMP, adenosine monophosphate; ATP, adenosine triphosphate; FAS, fatty acid synthase; FPLC, fast protein liquid chromatography; 3HQA, 3-hydroxyquinaldic acid; IPTG, isopropyl β-D-1-thiogalactopyranoside; MFabC, FabC cloned from Micromonospora sp. ML1; NADP+, nicotinamide adenine dinucleotide phosphate; NRPS, nonribosomal peptide


32

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

synthetase;

Ppant,

phosphopantetheinyl;

PPi,

pyrophosphate;

PPTase,

phosphopantetheinyltransferase; QXC, quinoxaline-2-carboxylic acid; RP-HPLC, reversed-phase high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SlFabC, FabC cloned from Streptomyces lasaliensis. REFERENCES [1] Perez Baz, J., Canedo, L. M., Fernandez Puentes, J. L., and Silva Elipe, M. V. (1997) Thiocoraline, a novel depsipeptide with antitumor activity produced by a marine Micromonospora. II. Physico-chemical properties and structure determination, J. Antibiot. 50, 738-741. [2] Romero, F., Espliego, F., Perez Baz, J., Garcia de Quesada, T., Gravalos, D., de la Calle, F., and Fernandez-Puentes, J. L. (1997) Thiocoraline, a new depsipeptide with antitumor activity produced by a marine Micromonospora. I. Taxonomy, fermentation, isolation, and biological activities, J. Antibiot. 50, 734-737. [3] Zolova, O. E., Mady, A. S., and Garneau-Tsodikova, S. (2010) Recent developments in bisintercalator natural products, Biopolymers 93, 777-790. [4] Okada, H., Suzuki, H., Yoshinari, T., Arakawa, H., Okura, A., Suda, H., Yamada, A., and Uemura, D. (1994) A new topoisomerase II inhibitor, BE-22179, produced by a streptomycete. I. Producing strain, fermentation, isolation and biological activity, J. Antibiot. 47, 129-135. [5] Matson, J. A., and Bush, J. A. (1989) Sandramycin, a novel antitumor antibiotic produced by a Nocardioides sp. Production, isolation, characterization and biological properties, J. Antibiot. 42, 1763-1767.


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[6] Takahashi, K., Koshino, H., Esumi, Y., Tsuda, E., and Kurosawa, K. (2001) SW-163C and E, novel antitumor depsipeptides produced by Streptomyces sp. II. Structure elucidation, J. Antibiot. 54, 622-627. [7] Toda, S., Sugawara, K., Nishiyama, Y., Ohbayashi, M., Ohkusa, N., Yamamoto, H., Konishi, M., and Oki, T. (1990) Quinaldopeptin, a novel antibiotic of the quinomycin family, J. Antibiot. 43, 796-808. [8] Konishi, M., Ohkuma, H., Sakai, F., Tsuno, T., Koshiyama, H., Naito, T., and Kawaguchi, H. (1981) BBM-928, a new antitumor antibiotic complex. III. Structure determination of BBM-928 A, B and C, J. Antibiot. 34, 148-159. [9] Yoshida, T., Katagiri, K., and Yokozawa, S. (1961) Studies on quinoxaline antibiotics. II. Isolation and properties of quinomycins A, B and C, J. Antibiot. 14, 330-334. [10] Shoji, J. I., and Katagiri, K. (1961) Studies on quinoxaline antibiotics. II. New antibiotics, triostins A, B and C, J. Antibiot. 14, 335-339. [11] Lingham, R. B., Hsu, A. H., O'Brien, J. A., Sigmund, J. M., Sanchez, M., Gagliardi, M. M., Heimbuch, B. K., Genilloud, O., Martin, I., Diez, M. T., Hirsch, C. F., Zink, D. L., Liesch, J. M., Koch, G. E., Gartner, S. E., Garrity, G. M., Tsou, N. N., and Salituro, G. M. (1996) Quinoxapeptins: novel chromodepsipeptide inhibitors of HIV-1 and HIV-2 reverse transcriptase. I. The producing organism and biological activity, J. Antibiot. 49, 253-259. [12] Erba, E., Bergamaschi, D., Ronzoni, S., Faretta, M., Taverna, S., Bonfanti, M., Catapano, C. V., Faircloth, G., Jimeno, J., and D'Incalci, M. (1999) Mode of action of thiocoraline, a natural marine compound with anti-tumour activity, Br. J. Cancer 80, 971-980. [13] Keller, U., and Schauwecker, F. (2001) Nonribosomal biosynthesis of microbial chromopeptides, Prog. Nucl. Acid. Res. Mol. Biol. 70, 233-289.


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Biochemistry

[14] Wyche, T. P., Dammalapati, A., Cho, H., Harrison, A. D., Kwon, G. S., Chen, H., Bugni, T. S., and Jaskula-Sztul, R. (2014) Thiocoraline activates the Notch pathway in carcinoids and reduces tumor progression in vivo, Cancer Gene Ther. 21, 518-525. [15] Taal, B. G., and Visser, O. (2004) Epidemiology of neuroendocrine tumours, Neuroendocrinology 80 Suppl 1, 3-7. [16] Mady, A. S., Zolova, O. E., Millan, M. A., Villamizar, G., de la Calle, F., Lombó, F., and Garneau-Tsodikova, S. (2011) Characterization of TioQ, a type II thioesterase from the thiocoraline biosynthetic cluster, Mol. Biosyst. 7, 1999-2011. [17] Sheoran, A., King, A., Velasco, A., Pero, J. M., and Garneau-Tsodikova, S. (2008) Characterization of TioF, a tryptophan 2,3-dioxygenase involved in 3-hydroxyquinaldic acid formation during thiocoraline biosynthesis, Mol. Biosyst. 4, 622-628. [18] Biswas, T., Zolova, O. E., Lombo, F., de la Calle, F., Salas, J. A., Tsodikov, O. V., and Garneau-Tsodikova, S. (2010) A new scaffold of an old protein fold ensures binding to the bisintercalator thiocoraline, J. Mol. Biol. 397, 495-507. [19] Al-Mestarihi, A. H., Villamizar, G., Fernandez, J., Zolova, O. E., Lombó, F., and GarneauTsodikova, S. (2014) Adenylation and S-methylation of cysteine by the bifunctional enzyme TioN in thiocoraline biosynthesis, J. Am. Chem. Soc. 136, 17350-17354. [20] Zolova, O. E., and Garneau-Tsodikova, S. (2012) Importance of the MbtH-like protein TioT in production and activation of the thiocoraline adenylation domain of TioK, MedChemComm 3, 950-955. [21] Lombó, F., Velasco, A., Castro, A., de la Calle, F., Brana, A. F., Sanchez-Puelles, J. M., Mendez, C., and Salas, J. A. (2006) Deciphering the biosynthesis pathway of the antitumor


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thiocoraline from a marine actinomycete and its expression in two streptomyces species, ChemBioChem 7, 366-376. [22] Reid, D. G., Doddrell, D. M., Williams, D. H., and Fox, K. R. (1984) A

15

N nuclear

magnetic resonance study of the biosynthesis of quinoxaline antibiotics, Biochim. Biophys. Acta 798, 111-114. [23] Yoshida, T., and Katagiri, K. (1969) Biosynthesis of the quinoxaline antibiotic, triostin, by Streptomyces s-2-210L, Biochemistry 8, 2645-2651. [24] Glund, K., Schlumbohm, W., Bapat, M., and Keller, U. (1990) Biosynthesis of quinoxaline antibiotics: purification and characterization of the quinoxaline-2-carboxylic acid activating enzyme from Streptomyces triostinicus, Biochemistry 29, 3522-3527. [25] Watanabe, K., Hotta, K., Praseuth, A. P., Koketsu, K., Migita, A., Boddy, C. N., Wang, C. C., Oguri, H., and Oikawa, H. (2006) Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli, Nat. Chem. Biol. 2, 423-428. [26] Praseuth, A. P., Wang, C. C., Watanabe, K., Hotta, K., Oguri, H., and Oikawa, H. (2008) Complete sequence of biosynthetic gene cluster responsible for producing triostin A and evaluation of quinomycin-type antibiotics from Streptomyces triostinicus, Biotechnol. Prog. 24, 1226-1231. [27] Watanabe, K., Hotta, K., Nakaya, M., Praseuth, A. P., Wang, C. C., Inada, D., Takahashi, K., Fukushi, E., Oguri, H., and Oikawa, H. (2009) Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic enzyme into echinomycin biosynthetic pathway for rational design and synthesis of potent antibiotic unnatural natural product, J. Am. Chem. Soc. 131, 9347-9353.


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[28] Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A. (1999) Practical Streptomyces genetics, John Innes Foundation. [29] Corpet, F. (1988) Multiple sequence alignment with hierarchical clustering, Nucl. Acids Res. 16, 10881-10890. [30] Riego, E., Bayo, N., Cuevas, C., Albericio, F., and Alvarez, M. (2005) A new approach to 3hydroxyquinoline-2-carboxylic acid, Tetrahedron 61, 1407-1411. [31] Stachelhaus, T., Mootz, H. D., and Marahiel, M. A. (1999) The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases, Chem. Biol. 6, 493-505. [32] Challis, G. L., Ravel, J., and Townsend, C. A. (2000) Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains, Chem. Biol. 7, 211-224. [33] Schmoock, G., Pfennig, F., Jewiarz, J., Schlumbohm, W., Laubinger, W., Schauwecker, F., and Keller, U. (2005) Functional cross-talk between fatty acid synthesis and nonribosomal peptide synthesis in quinoxaline antibiotic-producing streptomycetes, J. Biol. Chem. 280, 4339-4349. [34] Al-Mestarihi, A. H., Garzan, A., Kim, J. M., and Garneau-Tsodikova, S. (2015) Enzymatic evidence for a revised congocidine biosynthetic pathway, ChemBioChem 16, 1307-1313. [35] Zhang, W., Heemstra, J. R., Jr., Walsh, C. T., and Imker, H. J. (2010) Activation of the pacidamycin PacL adenylation domain by MbtH-like proteins, Biochemistry 49, 9946-9947. [36] Felnagle, E. A., Barkei, J. J., Park, H., Podevels, A. M., McMahon, M. D., Drott, D. W., and Thomas, M. G. (2010) MbtH-like proteins as integral components of bacterial nonribosomal peptide synthetases, Biochemistry 49, 8815-8817.


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[37] Zolova, O. E., and Garneau-Tsodikova, S. (2014) KtzJ-dependent serine activation and Omethylation by KtzH for kutznerides biosynthesis, J. Antibiot. 67, 59-64. [38] May, J. J., Kessler, N., Marahiel, M. A., and Stubbs, M. T. (2002) Crystal structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases, Proc. Natl. Acad. Sci., U. S. A. 99, 12120-12125. [39] Evans, B. S., Chen, Y., Metcalf, W. W., Zhao, H., and Kelleher, N. L. (2011) Directed evolution of the nonribosomal peptide synthetase AdmK generates new andrimid derivatives in vivo, Chem. Biol. 18, 601-607.


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Biochemistry

IMAGE for Table of Content: OH N

OH

TioJ

OH

O ATP 3-hydroxyquinaldic acid (1)

OAMP

N O

PP i Sfp, CoA

FabC FabC OH

THIOCORALINE

S

N O


39

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

TioK/TioT CO2H TioI TioQ NH 2 TioF N H L-Trp

Page 40 of 45

FabC OH

OH

TioJ

OH N TioL/M O ATP TioG 3-hydroxyquinaldic acid (1) TioH

FabC Sfp, CoA

O

PP i

OH

OAMP

N

S

N O

TioN/TioT TioR/TioT TioS/TioT MeS O O N

B

Ecm13/Ecm8 CO2H Ecm12 Ecm2 NH 2 Ecm11 N H L-Trp

Ecm14 Ecm4 Ecm3

O N

N S

N H OH

O

H N

S O O S

N H

S N

N

O

HO H N

O

N

O O

SMe

Thiocoraline FabC N OH

N O QXC (2)

N

Ecm1

FabC OAMP

N ATP

O

PP i

N Sfp, CoA

S

N O

Ecm6/Ecm8 Ecm7/Ecm8

O O O N N

N

N O N H

O

S H N

O S O

O

N H

O N

N O

Echinomycin


N H N

N O O

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Biochemistry

OH OH

N

OH

N 1 OH

OH

N

O

O

2 N OH

N OH

5

6

O

OH OH

OH

N

O

7

9

OH

10

OH

N

O

8

O

NH O

O

O N H

O

4 Br

H 2N N H

OH

N

O

3

N

O

OH

N

N H 11

OH

O N H 12

OH

OH

O NH O

NH

HN

O O N H 13

OH

N H 14

OH

NH

O HN

O N H 15

OH

N


16

O OH

Biochemistry

A 100

Relative % activity

80 60 40

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 L-Ala L-Arg L-Asn L-Asp L-Cys L-Gln L-Glu L-Gly L-His L-Ile L-Leu L-Lys L-Met L-Phe L-Pro L-Ser L-Thr L-Try L-Tyr L-Val

20

Substrate

B

a

b

c

d

100 Relative % activity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

80 60 40 20

ACS Paragon Plus Environment 0

1 2 3 4 5 6

1

1 2 3 4 5 6 Substrate

1 2 3 4 5 6

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Page 43 of 45

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Biochemistry

3HQA (1) std MFabC SlFabC TioO No T domain 21

22

23

24 25 Time (min)

26


27

28

Biochemistry

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3HQA (1) std 60 min 30 min 15 min 5 min 2 min 1 min 0.5 min 0 min 21

22

23

24 25 Time (min)

26

27

28


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Biochemistry

OH N

OH

TioJ

OH

O ATP 3-hydroxyquinaldic acid (1)

OAMP

N O

PP i Sfp, CoA

FabC FabC OH

THIOCORALINE

S

N O


Activation and loading of the starter unit during thiocoraline biosynthesis

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