PokMT1 from the Polyketomycin Biosynthetic Machinery of

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PokMT1 from the polyketomycin biosynthetic machinery of Streptomyces diastatochromogenes Tü6028 belongs to the emerging family of Cmethyltransferases that act on CoA-activated aromatic substrates Xun Guo, Ivana Crnovcic, Chin-Yuan Chang, Jun Luo, Jeremy R. Lohman, Monica Papinski, Andreas Bechthold, Geoffrey P Horsman, and Ben Shen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01219 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Biochemistry

PokMT1 from the polyketomycin biosynthetic machinery of Streptomyces diastatochromogenes Tü6028 belongs to the emerging family of C-methyltransferases that act on CoA-activated aromatic substrates

Xun Guo,1,# Ivana Crnovcic,1,# Chin-Yuan Chang,1 Jun Luo,1 Jeremy R. Lohman,1 Monica Papinski,2 Andreas Bechthold,3 Geoffrey P. Horsman,2 and Ben Shen*1,4,5*

1

Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458, USA;

2

Department of Chemistry and Biochemistry, Wilfrid Laurier University, Waterloo, ON N2L 3C5, Canada; 3Institute for Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, University of Freiburg, Stefan-Meier-Str. 19, 79104 Freiburg, Germany; 4Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33458, USA; and 5Natural Products Library Initiative at The Scripps Research Institute, The Scripps Research Institute, Jupiter, FL 33458, USA

*To whom correspondence should be addressed: The Scripps Research Institute, 130 Scripps Way, #3A1, Jupiter, FL 33458; Tel: (561) 228-2456; Email: [email protected]

Running title: PokMT1 C-MT acting on a CoA-activated substrate

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ACS Paragon Plus Environment

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ABSTRACT Recent

biochemical

characterizations

of

the

MdpB2

CoA

ligase

and

MdpB1

C-

methyltransferase (C-MT) from the maduropeptin (MDP, 2) biosynthetic machinery revealed unusual pathway logic involving C-methylation occurring on a CoA-activated aromatic substrate. Here we confirmed this pathway logic for the biosynthesis of polyketomycin (POK, 3). Biochemical characterization unambiguously established that PokM3 and PokMT1 catalyze the sequential conversion of 6-methylsalicylic acid (6-MSA, 4) to form 3,6-dimethylsalicylyl-CoA (3,6-DMSA-CoA, 6), which serves as the direct precursor for the 3,6-dimethylsalicylic acid (3,6DMSA) moiety in the biosynthesis of 3.

PokMT1 catalyzes the C-methylation of 6-

methylsalicylyl-CoA (6-MSA-CoA, 5) with a kcat of 1.9 min-1 and a Km of 2.2 ± 0.1 M, representing the most proficient C-MT characterized to date. Bioinformatics analysis of MTs from natural product biosynthetic machineries demonstrated that PokMT1 and MdpB1 belong to a phylogenetic clade of C-MTs that preferably act on aromatic acids. Significantly, this clade includes the structurally characterized enzyme SibL, which catalyzes C-methylation of 3hydroxykynurenine in its free acid form, using two conserved tyrosine residues for catalysis. A homology model and site-directed mutagenesis suggested that PokMT1 also employs this unusual arrangement of tyrosine residues to coordinate C-methylation but revealed a large cavity capable of accommodating the CoA moiety tethered to 5. CoA-activation of the aromatic acid substrate may represent a general strategy that could be exploited to improve catalytic efficiency. This study sets the stage to further investigate and exploit the catalytic utility of this emerging family of C-MTs in biocatalysis and synthetic biology.

Keywords: maduropeptin, polyketomycin, methyltransferase, 6-methylsalicylic acid

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Biochemistry

INTRODUCTION Biosynthesis of natural products often involves the elaboration of a core scaffold (e.g., a polyketide or nonribosomal peptide) with various peripheral moieties.1

This biosynthetic

pathway logic is exemplified by the convergent biosynthesis of enediynes such as neocarzinostatin (NCS, 1), where an aminosugar and aromatic acid decorate the enediyne polyketide core (Figure 1A).2 The biological activation and transfer of acyl groups often employ thiols like coenzyme A (CoA) to generate reactive thioesters that can be appended to a nucleophilic acceptor on the core scaffold. For example, the naphthoyl moiety of 1 and the 3,6dimethylsalicylic acid (3,6-DMSA) moiety of maduropeptin (MDP, 2) are installed on their respective polyketide scaffolds as activated CoA thioesters (Figure 1B).

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Figure 1. (A) Chemical structure of NCS (1), MDP (2), and POK (3) with the aromatic acid moieties highlighted in red; (B) Convergent strategies for natural product biosynthesis featuring a common pathway logic as exemplified by NcsB and NcsB1 that biosynthesize the full modified naphthoic acid moiety prior its activation by NcsB2 for the eventual incorporation into 2 (path a) or an unusual pathway logic as exemplified by MdpB2/PokM3 that activate 4 followed by MdpB1/PokMT1 that catalyze C-methylation of a CoA-activated substrate 5 before its eventual incorporation into 3 (path b). Elucidation of the biosynthetic pathways for the naphthoic acid and 3,6-DMSA moieties of 1 and 2, respectively, revealed unexpected timing for the CoA activation step of the latter. In the case of 1, the NcsB type I iterative polyketide synthase (PKS) assembles the naphthoic acid scaffold, which is sequentially hydroxylated by the NcsB3 hydroxylase and methylated by the NcsB1 Omethyltransferase (O-MT). As expected, the resulting fully modified naphthoic acid moiety is activated as a CoA thioester by the NcsB2 CoA ligase prior to its installation onto the NCS

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Biochemistry

enediyne core (Figure 1B, path a).2–5 In contrast, the 3,6-DMSA moiety of 2 is not fully modified prior to CoA activation. Instead, 6-methylsalicylic acid (6-MSA, 4), synthesized by the MdpB iterative type I PKS, is activated by MdpB2 to generate 6-methylsalicylyl-CoA (6-MSA-CoA, 5) first, followed by MdpB1-catalyzed C-methylation to form 3,6-dimethylsalicylyl-CoA (3,6-DMSACoA, 6) (Figure 1B, path b).6,7

Acyl activation as a CoA thioester occurring prior to C-

methylation represents a surprising pathway timing that significantly diverges from the established and intuitive pathway logic of CoA activation immediately prior to installation on a core scaffold as exemplified in the biosynthesis of 1 (Figure 1B, path a).

Significantly, this unusual pathway logic revealed MdpB1 as a member of an apparently novel family of C-methyltransferases (C-MTs). MdpB1 C-methylated CoA-activated 5 and possessed no activity towards the free acid 4. This unusual specificity for a CoA-activated substrate has previously been observed only for the caffeoyl-CoA O-MT (PfoMT),8 although other O-MTs such as CalO1 and CalO6 have been proposed to act on CoA- or ACP-tethered substrates based on their preference for N-acetylcysteamine thioesters as substrate mimics.9,10 However, detailed kinetic analyses and exploration of substrate scope were impaired by several technical challenges associated with MdpB1,7 prompting us to search for a homologous C-MT as a model system for in-depth mechanistic investigations.

Polyketomycin (POK, 3), a tetracyclic quinine glycoside antitumor antibiotic isolated from Streptomyces diastatochromogenes Tü6028, possesses the same 3,6-DMSA moiety as 2 (Figure 1A). The biosynthetic gene cluster for 3 has been characterized, featuring PokM3 and PokMT1, which possess 73% and 72% sequence identity to MdpB2 and MdpB1 and are predicted to be a CoA ligase and C-MT, respectively.11–13 This striking similarity implies that a common pathway logic may be employed to furnish the 3,6-DMSA moieties in the biosynthesis

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of both 2 and 3 (Figure 1B, path b). Here we report the biochemical characterization of PokM3 and PokMT1.

PokM3 catalyzed ATP-dependent ligation of CoA and 4 to afford the CoA-

activated thioester 5, and PokMT1 catalyzed S-adenosylmethionine (SAM)-dependent Cmethylation of 5 to form thioester 6, setting the stage to better understand the chemistry underlying the unusual C-methylation of CoA activated aromatic substrates.

MATERIALS AND METHODS Cloning, Overproduction, and Purification of PokM3 and PokMT1. Strains, plasmids and primers used in this study are listed in Tables S1, S2 and S3, respectively. The pokM3 and pokMT1 genes were PCR-amplified from cosmid CB30-6D2013 using Platinum Pfx DNA polymerase from Invitrogen (Carlsbad, CA) and the following primers: PokM3-LIC-F, PokM3LIC-R, PokMT1-LIC-F and PokMT1-LIC-R (Table S3). The purified PCR products were treated with T4 DNA polymerase/dCTP and directly cloned into the pBS308014 vector treated with BsmFI and T4 DNA polymerase/dGTP successively to generate plasmids pBS10020 and pBS10021 (Table S2), which were sequenced to confirm PCR fidelity.

For site-directed

mutagenesis of pokMT1, the pokMT1 gene from pBS10021 was amplified by PCR with Q5 DNA polymerase (NEB) in two steps by primer extension15 using the PokMT1-LIC-F and PokMT1LIC-R primers with internal primers containing the desired mutation(s) (Table S3). The mutant pokMT1 genes were then cloned into pBS3080 as described above yielding pBS10022– pBS10026 (Table S2).

Plasmids pBS10020 and pBS10021, encoding the N-terminal His6-tagged PokM3 and PokMT1 respectively, were transformed into chemically competent E. coli BL21(DE3) cells. The resulting transformants were grown in LB medium, supplemented with 50 g/mL kanamycin, to OD600 of 0.6. Gene expression was induced by adding 0.4 mM IPTG and incubating overnight at 18 °C. The cells from 1 L of culture were harvested and lysed in 30 mL of lysis buffer (100 mM Tris, 6


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Biochemistry

300 mM NaCl, 15 mM imidazole, 10% glycerol, 1 mM benzamidine, 2 mg/L leupepsin, 2 mg/L pepstatin, 5 mM -mercaptoethanol, pH 8.0) by two passages through a French pressure cell at 10,000 psi. The resulting cell lysate was centrifuged at 53,000 x g to remove cell debris, and the filtered supernatant from 0.45 m syringe filter was loaded onto a pre-equilibrated HisTrap FF 5 mL Ni-NTA agarose column with chilled buffer A (50 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 8.0). After being washed with 10 volumes of buffer A, PokM3 was eluted with 5 volumes of buffer B (50 mM Tris, 100 mM NaCl, 500 mM imidazole, pH 8.0). The proteincontaining eluent was concentrated with a 30K MWCO ultrafiltration device to a volume of 2.5 mL, prior to desalting over a PD-10 column into storage buffer (50 mM Tris, 50 mM NaCl, pH 8.0). Aliquots were concentrated, flash-frozen in liquid nitrogen, and stored at -80 °C until use. Each of the PokMT1 site-directed mutants was produced and purified as described above. The molecular weight (MW) and oligomeric state of PokM3, PokMT1, and MdpB1 in solution was determined by analytical size-exclusion chromatography using a HiLoad Superdex 200 pg (16 × 600 mm) column (GE Healthcare Life Sciences, PA) connected to a FPLC system (GE Healthcare Life Sciences, PA). The column was pre-equilibrated with two column volumes of storage buffer (50 mM Tris, pH 8.0) and calibrated with carbonic anhydrase (29 kDa), ovalbumin (44 kDa), conalbumin (75 kDa), alcohol dehydrogenase (150 kDa), and β-amylase (200 kDa). The chromatography was carried out at 4 °C at a flow rate of 1 mL min−1. The calibration curve of Kav versus log(MW) was prepared using the equation Kav = Ve − Vo/(Vt −Vo), where Ve, Vo, and Vt is the elution volume, column void volume, and total bed volume, respectively.

PokM3-Catalyzed Ligation Reaction between 6-MSA (4) and CoA. In a total volume of 200 L of assay buffer (100 mM HEPES, 100 mM NaCl, 1 mM EDTA, 10 mM MgCl2, pH 7.40), PokM3 (20 M) was incubated with ATP (4 mM), CoA (1 mM), 4 (0.5 mM), BSA (0.1 mg/mL), and TCEP (0.1 mM) at 30 °C for 10 min. The reaction was then quenched by 2.0 M HCl (10 L),

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followed by removal of the precipitate by centrifugation for 3 min at 15,700 x g. The clarified supernatant was directly subjected to HPLC analysis with UV detection.

Controls without

enzyme or CoA were also performed. The PokM3-catalyzed ligation reaction of 4 was scaled up, and the CoA thioester product 5 was purified twice by HPLC using an Altima C18 reverse phase column (250 x 10 mm, 5 m; Grace Davison, Deerfield, IL) with the same linear gradients as the analysis method at a flow rate of 5.0 mL/min. After removal of the solvent by rotary evaporation, the residue was lyophilized overnight and subjected to ESI-MS analysis in both negative and positive ion modes.

6-MSA-CoA (5): ESI-MS affording an [M+H]+ ion at m/z 902.4 and an [M-H]- ion at m/z 900.6, respectively, consistent with the molecular formula C29H42N7O18P3S (calcd for the [M+H]+ ion at m/z 902.2 and the [M-H]- ion at m/z 900.1).

PokMT1 Catalyzed C-Methylation of 6-MSA-CoA (5) to form 3,6-DMSA-CoA (6). In a total volume of 100 L of assay buffer (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.50), PokMT1 (20 M) was incubated with SAM (1 mM), TCEP (0.1 mM), and 5 (0.5 mM) at 30 °C for 40 min. The reaction was then quenched with 2.0 M HCl (4 L) followed by removal of the precipitate by centrifugation. The clarified supernatant was subjected to HPLC analysis, using the same linear gradient as PokM3, with UV detection. Controls without enzyme or SAM were also performed. In addition, a negative control with denatured PokMT1 (i.e., boiled for 5 min) was also performed. The PokMT1-catalyzed methylation reaction of 5 was scaled up and the resultant product 6 was purified twice by HPLC using an Altima C18 reverse phase column (250 x 10 mm, 5 m; Grace Davison, Deerfield, IL) with the same linear gradients as the analysis method at a flow rate of 5.0 mL/min. After removal of the solvent by rotary evaporation, the residue was lyophilized overnight and subjected to high-resolution ESI-MS analysis (Figure S1)

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Biochemistry

in positive ion mode.

Structural assignment of 6 as the product of PokMT1-catalyzed

methylation of 5 was further supported by 1H and 13C NMR analysis (Figure S2).

To test the free acid 6-MSA (4) as an alternative substrate for PokMT1, in a total volume of 100 L of assay buffer (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.50), PokMT1 (20 M) was incubated with SAM (1 mM), TCEP (0.1 mM), and 4 (0.5 mM) at 30 °C for 1 h. The reaction was then quenched with 2.0 M HCl (4 L) followed by removal of the precipitate by centrifugation. The clarified supernatant was subjected to HPLC analysis, using the same linear gradient as PokM3, with UV detection.

To carry out PokM3 and PokMT1-catalyzed one-pot transformation of the free acid 6-MSA (4) into the methylated CoA thioester 3,6-DMSA-CoA (6), in a total volume of 200 L of assay buffer (100 mM HEPES, 100 mM NaCl, 1 mM EDTA, 10 mM MgCl2, pH 7.40), PokM3 (20 M), and PokMT1 (20 M) were incubated with ATP (4 mM), CoA (1 mM), SAM (1 mM), BSA (0.1 mg/mL), TCEP (0.1 mM), and 4 (0.5 mM) at 30 °C for 1 h. The reaction was then quenched with 2.0 M HCl (10 L) followed by removal of the precipitate by centrifugation for 3 min at 15,700 x g. The clarified supernatant was directly subjected to HPLC analysis, using the same linear gradient as PokM3, with UV detection.

3,6-DMSA-CoA (6): HR ESI-MS affording an [M+H]+ ion at m/z 916.1738, consistent with the molecular formula C30H44N7O18P3S (calcd for the [M+H]+ ion at m/z 916.1749). 1H NMR (DMSOd6, 400 MHz): 8.63 (s, 1H, H-6), 8.39 (s, 1H, H-2), 8.12 (t, J = 5.6 Hz, 1H, NH), 7.72 (t, J = 5.6 Hz, 1H, NH), 6.98 (d, J = 7.4 Hz, 1H, H-26), 6.58 (d, J = 7.4 Hz, 1H, H-27), 5.94 (d, J = 5.6 Hz, 1H, H-8), 4.73 (m, 1H, H-9), 4.67 (m, 1H, H-11), 4.37 (s, 1H, H-15), 4.16 (m, 2H,H-12), 3.86 (m, 1H, H-13), 3.67 (s, 1H, H-10), 3.57 (m, 1H, H-13), 3.21 (m, 4H, H-17,20), 3.01 (t, J = 7.0 Hz, H-

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21), 2.24 (t, J = 7.0 Hz, H-18), 2.07 (s, 6H, H-29,30), 0.85 (s, 3H, H-30), 0.70 (s, 3H, H-31).

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13

C

NMR (DMSO-d6, 100 MHz): 194.7 (C-22), 172.1 (C-16), 170.7 (C-19), 158.7 (C-4), 158.4 (C-24), 150.95 (C-2), 148.4 (C-6), 141.5 (C-7a), 131.9 (C-28), 131.7 (C-26), 128.9 (C-23), 123.0 (C-27), 118.6 (C-4a), 118.6 (C-25), 87.3 (C-8), 82.0 (C-11), 74.0 (C-9), 73.4 (C-10), 72.9 (C-13), 72.3 (C-15), 65.0 (C-12), 38.2 (C018), 35.1 (C-20), 34.8 (C-17), 30.6 (C-14), 28.6 (C-21), 20.9 (C-31), 19.0 (C-32), 18.2 (C-29), 16.0 (C-30).

PokM3 and PokMT1 Steady-State Kinetics. The steady-state kinetic constants of PokM3catalyzed formation of the CoA thioester 5 were determined using the single-point methodology.16

Preliminary experiments established that the rates for enzyme-catalyzed

formation of 5 from 4 under the assay conditions examined were linear over a period of 4 min. All single-point kinetic assays were therefore based on an incubation time of 45 s. Eight assays with a total volume of 500 L in assay buffer (100 mM HEPES, 100 mM NaCl, 1 mM EDTA, 10 mM MgCl2, pH 7.40) containing ATP (2 mM), CoA (2 mM), BSA (0.1 mg/mL), TCEP (0.1 mM), and 4 at variable final concentrations (1, 1.5, 2, 2.5, 3, 3.5, 4, 8.33 M) were pre-incubated at 30 °C for 3 min. The reactions were initiated by adding 0.61 g of PokM3 (0.01 nmol, 20 nM final concentration) and incubated at 30 °C for 45 s followed by quenching with 25 L of 2.0 M HCl. The quenched reaction mixtures were then lyophilized overnight and re-dissolved with 20 L of deionized water before HPLC analysis, using the same linear gradient as used for the PokM3 activity assay, with UV detection. Each reaction was repeated using different protein preparations. A standard curve of enzymatically prepared 5 (0.1, 0.2, 0.3, 0.4, 0.5 nmol) was employed for quantification.

Steady-state kinetic constants were extracted by fitting the

Michaelis-Menten equation to the data by non-linear regression using the open source statistical package RStudio version 1.0.136.17

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Biochemistry

The steady-state kinetic constants for PokMT1-catalyzed C-methylation of 5 to form thioester 6 were similarly determined using the single-point methodology.16 A time course of PokMT1catalyzed formation of 3,6-DMSA-CoA (6) from 5 and SAM was monitored, establishing that the reaction rates under the assay conditions examined were linear over a period of at least 8 min. Assay mixtures of 500 µL, containing SAM (1 mM), TCEP (0.2 mM), and 5 (2, 3, 4, 5, 6, 7, 8, 9, 10 µM) in assay buffer (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 12 mM MgCl2, pH 7.50), were pre-heated at 30 °C for 3 min followed by addition of 200 nM of PokMT1 to initiate the reaction. The reaction was allowed to continue at 30 °C for 4 min prior to being quenched with 20 µL of 2.0 M HCl, frozen in liquid nitrogen, and lyophilized to dryness. The reaction residues were then dissolved in 20 µL of deionized water and analyzed by HPLC as described above. The experiment was repeated in duplicate on two different days to generate 18 total initial rate measurements. A standard curve of enzymatically prepared 6 was employed for quantification. Steady-state kinetic constants were extracted by fitting the Michaelis-Menten equation to the data by non-linear regression using the open source statistical package RStudio version 1.0.136.17

For the PokMT1 site-directed mutants relative activities were determined as

described above using a 6-MSA-CoA (5) concentration of 0.5 mM. All reactions were performed in triplicate.

Construction of Phylogenetic Tree of selected MTs. C-, N- and O-MTs were screened by a BLASTp search, and the amino acid sequences (see SI for accession numbers) were aligned for comparison. The phylogenetic tree was generated using Clustal Omega18 and visualized using Hypertree.19

Homology Modeling of PokMT1 and MdpB1. The primary amino acid sequences of PokMT1 and

MdpB1

were

submitted

to

the

online

I-TASSER

server

(http://zhanglab.ccmb.med.umich.edu/I-TASSER) to generate homology models by iterative 11


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threading assembly.20

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The coordinates of the resulting top-ranked models were visualized

using VMD and PyMOL.21,22 The volume of the active site cavity was calculated by POCASA.23

RESULTS Cloning, Overproduction, and Purification of PokM3 and PokMT1. PokM3, PokMT1 and PokMT1 site-directed mutants were overproduced in E. coli as N-terminal His6-tagged fusion proteins and purified to homogeneity by Ni-NTA affinity chromatography. The yield of purified PokM3 was 20 mg per liter of culture at >95% purity by SDS-PAGE, with an apparent molecular weight consistent with the expected value of 61,303 Da (Figure S3A). The PokMT1 purification yielded 33 mg/L at >99% purity by SDS-PAGE, migrating as expected for the predicted molecular weight of 41,144 Da (Figure S3A). The yields and the purity measured by SDSPAGE (Figure S3B) of PokMT1 mutants were comparable to the wild-type enzyme.

As

determined by size exclusion chromatography PokM3 and PokMT1 form a dimer and a tetramer in solution, respectively (Figure S4).

PokM3-Catalyzed Formation of 6-MSA-CoA (5). The catalytic activity of PokM3 was assayed in the presence of the cognate substrates 4, CoA, and ATP in HEPES buffer, pH 7.4, supplemented with 10 mM MgCl2. PokM3 was shown to be catalytically active, generating 5 with retention time at 9.2 min (Figure 2A, panel II). As exemplified by MdpB2,7 the PokM3 catalyzed enzymatic reaction started with activation of 4 (retention time 8.9 min) by ATP (retention time 1.5 min) to form the 4-O-AMP intermediate, followed by conversion into 5 upon nucleophilic attack of the free thiol of CoA (retention time 2.2 min). The control assays without CoA or PokM3 showed only unmodified 4 as expected (Figure 2A, panel I). This enzymatic reaction was scaled up, and the resultant CoA thioester product 5 was purified twice by HPLC and confirmed by ESI-MS.

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Biochemistry

Figure 2. In vitro assays of PokM3 as a CoA ligase and PokMT1 as a C-MT. (A) HPLC analysis of PokM3-catalyzed ligation between 6-MSA (4) and CoA in the presence of ATP and MgCl2: (I) control without PokM3; (II) complete assay. (B) HPLC analysis of PokMT1-catalyzed C-methylation of 6-MSA-CoA (5) in the presence of SAM: (I) control without PokMT1; (II) control with denatured PokMT1; (III) complete assay; (IV) coupled assay of PokM3 and PokMT1 transforming 6-MSA (4) to 3,6-DMSA (6) in the presence of ATP, MgCl2, and SAM. Catalytic Activity and Substrate Specificity of PokMT1. The catalytic activity of PokMT1 was investigated with enzymatically-generated and HPLC-purified CoA thioester 5 as a substrate in the presence of SAM in HEPES buffer, pH 7.5. The enzymatic reaction afforded 13


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the methylated thioester 6 as the sole product with retention time at 10.6 min, while the retention time of SAM was 2.1 min (Figure 2B, panel III).

Control assays without PokMT1 or SAM

showed no activity as expected (Figure 2B, panel I). PokMT1 was readily denatured upon boiling for 5 min or lowering the pH values below 5, and the resultant enzyme completely lost its activity to catalyze the formation of 6 (Figure 2B, panel II). Thus, in contrast to MdpB1,7 the ability to stop the PokMT1 reaction by heat or acidification makes it a good candidate for steadystate kinetic analysis. The enzymatic reaction was scaled up, and the resultant CoA thioester product 6 was purified by HPLC, the identity of which was confirmed by HR-ESI-MS, 1H, and 13C NMR spectroscopic analyses. The activity of PokMT1 was also tested with the free acid 4 as an alternative substrate, however, no formation of 6 was detected (Figure S5).

In addition,

PokMT1 can work with PokM3 in a one-pot conversion of 4 to 6 in the presence of all the required reactants (Figure 2B, panel IV), establishing their respective roles during the biosynthesis of 3 (Figure 1B, path b). Finally, the absence of any effect on the activity of PokMT1 upon the addition of 1 mM EDTA indicates that PokMT1 does not require magnesium or other divalent cations for catalysis.

Steady-State Kinetics of PokM3-catalyzed Formation of 6-MSA-CoA (5). The steady-state kinetic parameters for the PokM3-catalyzed formation of 5 were determined with saturating concentrations of CoA (2 mM) and ATP (2 mM). The formation of 5 followed Michaelis-Menten kinetics with a Km value of 1.3 ± 0.3 M for 4 and a kcat value of 0.45 ± 0.04 s-1 (Figure S6A).

Steady-State Kinetics of PokMT1-Catalyzed Formation of 3,6-DMSA-CoA (6). The steadystate kinetic parameters for the PokMT1-catalyzed formation of 6 were similarly determined with saturating concentrations of SAM (1 mM).

The formation of 6 followed Michaelis-Menten

14


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Biochemistry

kinetics with a Km value of 2.2 ± 0.1 M for 5 and a kcat value of (3.2 ± 0.1) x 10-2 s-1 (Figure S6B).

Phylogenetic analysis. Amino acid sequences of selected O-, C-, and N-MTs associated with natural product biosynthesis were used to construct a phylogenetic tree (Figure S7). With the exception of a small clade of class III MTs, the analysis was restricted primarily to bacterial class I SAM-dependent natural product MTs.24 Interestingly, although PfoMT is the only other MT known to catalyze O-methylation of a CoA-activated aromatic acid substrate,8 it belongs to a clade of metal-dependent MTs that are distantly related to MdpB1 and PokMT1 (Figure S7). Significantly, we identified a clade of aromatic acid C-MTs containing MdpB1 and PokMT1 as well as the well-characterized enzymes SacF, SfmM2, AcmI, AcmL, SibL, and Orf19.25–27

Structural Analysis of PokMT1 and MdpB1. In contrast to SibL, a related aromatic acid C-MT from Streptosporangium sibiricum,27 which forms a dimer in solution,28 PokMT1 and MdpB1 are tetramers in solution as determined by size exclusion chromatography (Figure S4).

The

homology models of PokMT1 and MdpB1 generated by I-TASSER were visualized using PyMOL and POCASA to evaluate the CoA-binding capacity of the active site. Importantly, a crystal structure is available of the ternary complex of SibL, which includes SAH and the substrate 3-hydroxykynurenine (3HK).27 Structural overlay of the PokMT1 and MdpB1 models with SibL revealed good fits, with RMSD values of 0.66 and 0.69 Å, respectively. The high similarities of the PokMT1 and MdpB1 models to the SibL structure provided an opportunity to compare volumes of the substrate binding sites using the surface cavity feature in PyMOL (Figure 3). Although the binding conformations for PokMT1 and MdpB1 were not modeled, the PokMT1 and MdpB1 internal binding pockets are qualitatively much larger (~2463 Å and ~2697 Å) than that of SibL (~1676Å), consistent with PokMT1 and MdpB1 acting on CoA-thioester 5 as the preferred substrate. The structure of SibL reveals that a tyrosine, which is quite conserved 15


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Page 16 of 30

in methyltransferases (Figure S8), disrupts the substrate binding cavity. Instead of the tyrosine in SibL, PokMT1 and MdpB1 contain in that position a serine residue, which may extend the substrate binding cavity to generate the enlarged space for the accommodation of CoA. The homology models of PokMT1 and MdpB1 also revealed a conserved Arg (Arg15 in PokMT1 or Arg16 in MdpB1, Figure S8) near the putative CoA binding cavity (Figure 3). PokMT1 mutant Arg15Ala showed significantly decreased activity of 97% (Table 3), suggesting that it may play a role in substrate binding.

Figure 3. Comparison of the active sites of SibL, PokMT1, and MdpB1 with interior cavities shown in gray surface representation. (A) The SibL:SAH:3HK ternary complex (PDB 4U1Q)27; Homology models of (B) PokMT1 and (C) MdpB1 including, for reference, SAH and 3HK coordinates from SibL generated after superimposing the two structures, and highlighting the larger cavities that could accommodate the CoA-activated substrate 6-MSA-CoA (5).

Many methyltransferases that have been structurally and biochemically characterized like SibL (Figure S9A-C) use a general acid-base mechanism in the active site for catalysis.27

Our

mechanistic proposal for PokMT1 includes Tyr140, Tyr301, Asn156, and Glu254 as possible catalytic residues (Figure 4 and Figure S9D-E).

All PokMT1 mutants remained to be

catalytically competent to transform 6-MSA-CoA (5) to 3,6-DMSA-CoA (6), albeit at varying activities (Table 3). The Tyr140Phe, Tyr301Phe, and Tyr140Phe-Tyr301Phe mutants showed significantly decreased activity of 97%, 80% and 97%, respectively, indicating that Tyr140F in particular, like in the case of SibL,27 is essential for substrate binding and/or catalysis.

In

comparison to Tyr140 and Tyr301 the residues Asn156 and Glu254 are unique to PokMT1 and MdpB1. Analysis of the PokMT1 mutants Asn156Ala and Glu254Ala showed decreased activity 16


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Biochemistry

of 67% and 96%, respectively. This data indicates that Asn156 may act as a stabilizer for Tyr301 and that the residue Glu254 is important for general base catalyzed rearomatization (Figure 4).

Figure 4. Proposed mechanism of PokMT1 with conserved active site residues. Tyr140 and Tyr301 are conserved among all aromatic acid C-MTs, while Asn156 and Glu254 are unique to the subgroup (PokMT1 and MdpB1) acting on CoA-activated substrates. DISCUSSION In our continued efforts to study enediyne biosynthesis, we recently discovered an unusual pathway logic from the biosynthetic machinery of 2, featuring MdpB1 as the first and only C-MT known to date that catalyzed C-methylation exclusively of a CoA-activated substrate (Figure 1B, path b)6,7

MdpB1 represents a growing family of novel MTs, in-depth mechanistic

characterization of which, however, was impaired by several technical challenges associated with MdpB1.7

POK (3) possesses a similar 3,6-DMSA moiety as MDP (2) (Figure 1A).

Comparative analysis of the MDP and POK biosynthetic machineries revealed that PokM3 and PokMT1 were homologues of MdpB2 and MdpB1, respectively, implying that the same pathway logic may be employed in the biosynthesis of 3 (Figure 1B, path b). Specifically, 73% identity between the MdpB2 and PokM3 CoA ligases, and 72% identity between the MdpB1 and PokMT1 C-MTs strongly implied identical functions, prompting us to consider PokM3 and PokMT1 as an alternative model system to study this emerging family of novel C-MTs. Indeed, PokM3 catalyzes ligation of CoA and 4 to afford 5 (Figure 1B, path b), with steady-state kinetic parameters within the normal range for CoA ligases acting on aromatic substrates (Table 1).7,29–

17


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33

Page 18 of 30

PokMT1 acts exclusively on the CoA-activated thioester 5 to form 6 (Figure 1B, path b),

representing the most proficient C-MT that has been characterized to date (Table 2).8,26,34 CoAactivation of the aromatic acid substrate may represent a general strategy that could be exploited to improve catalytic efficiency.

Although previous work revealed that the S.

diastatochromogenes pokMT1 mutant accumulated both POK-MD1 (7), which lacked the 3,6DMSA moiety, and POK-MD2 (8), the desmethyl congener of 3 (Figure S10A),13 PokMT1 was proposed to methylate 4, prior to activation of the resultant product 3,6-DMSA as an AMPtethered intermediate for its eventual incorporation into 3 (Figure S10B). This proposal should now be revised in light of the new findings from biochemical characterizations of PokM3 and PokMT1 in the current study (Figure 1B, path b). This biosynthetic pathway logic was further supported by the one-pot stoichiometric conversion of 4 to 6 in the presence of both PokM3 and PokMT1 (Figure 2B, panel IV).

A phylogenetic tree was constructed to obtain mechanistic insight into the unique class of CMTs acting on CoA-activated aromatic acids, exemplified by MdpB1 and PokMT1 (Figure S7). A comparison of MTs primarily from microbial natural product biosynthetic pathways24 revealed wide distribution of O-, N-, and C-MTs with minimal apparent phylogenetic clustering with respect to methylation atom type or substrate. For example, PfoMT, the metal-dependent plant O-MT and the only other MT confirmed to act on a CoA-tethered substrate,8 is relatively distantly related to MdpB1 and PokMT1. In contrast, the latter two enzymes are clustered adjacent to a family of enzymes that have been characterized to catalyze C-methylation of the aromatic ring of amino acids, such as tyrosine (SacF, SfmM2)25,35–37 and 3-hydroxykynurenine (AcmI, AcmL, Orf19, SibL).25–27 Using the eight sequences comprising the clade of aromatic acid C-MTs (Figure S7), we construct a more limited sequence alignment to identify potential active site amino acids (Figure S8). Together with the alignment, the crystal structure of SibL from Streptosporangium sibiricum in complex with SAH and 3HK critically informed mechanistic 18


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Biochemistry

hypotheses.27 Specifically, two conserved tyrosine residues, Tyr134 and Tyr295 (Figure S8), were observed within hydrogen bonding distance (~2.7 Å) of the 3-hydroxyl group of 3HK in the SibL active site.

Although steady-state kinetic analyses were not performed, activity

measurements revealed decreases of 98% and 44% for Y134F and Y295F mutations, respectively, indicating that Tyr134 in particular was important for catalysis.27

Based on the general acid-base mechanism proposed for SibL involving the conserved residues Tyr134 and Tyr295 and our own side-directed mutagenesis data, we can propose a similar mechanism and identify candidate catalytic residues for PokMT1 and MdpB1.

The SibL

mechanism was proposed to start with deprotonation of Tyr134 by Asp132, which results in a conformational change that displaces tyrosinate into the active site.27 After Tyr295-catalyzed methylation, the Tyr134 tyrosinate could catalyze -keto deprotonation to rearomatize the substrate. Interestingly, another conserved residue Tyr99 is within hydrogen bonding distance of the substrate amino moiety and may play a role in catalysis (Figure S9A-C).

Although

PokMT1 and MdpB1 also possess the conserved Tyr99, the homology models place this residue outside of the active site but maintain similar active site architecture of the conserved Tyr140 and Tyr301 residues (PokMT1 numbering; analogous to Tyr134 and Tyr295 in SibL). The equivalent of Asp132 in SibL is absent in PokMT1 and MdpB1, suggesting that both tyrosines are protonated at the beginning of the catalytic cycle. Among enzymes in the C-MT clade, PokMT1 and MdpB1 uniquely possess Asn156 and Glu254 as potential catalytic residues. For example, Asn156 may activate or stabilize Tyr301 just as the substrate amino group may interact with Tyr99 to stabilize Tyr295 in the SibL-catalyzed reaction (Figure S9D). In addition, the site-directed mutagenesis data and the model imply that the unique Glu254 residue is appropriately positioned for general base catalyzed rearomatization (Figure S9E). Overall, our mechanistic proposal supported by the site-directed mutagenesis data includes a larger pocket

19


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Page 20 of 30

to accommodate a CoA-tethered substrate, and Tyr140, Tyr301, Gln156, and Glu254 as important catalytic residues (Figure 4, Table 3).

In summary, we have characterized PokM3 as a CoA ligase and PokMT1 as a new member of the emerging family of novel C-MTs that act on CoA-activated aromatic substrates, establishing their timing in the biosynthesis of the 3,6-DMSA moiety of 3. Together with our early studies of MdpB1 and MdpB2 in the biosynthesis of 2, these findings highlight an unusual pathway logic for natural product biosynthesis that may be underappreciated. PokMT1 represents the most proficient C-MT that has been characterized to date, and CoA-activation of the aromatic acid substrate may represent a general strategy that could be exploited to improve catalytic efficiency. Informed by the crystal structure of the SibL:SAH:3HK ternary complex, multiple sequence alignment, homology modeling, and site-directed mutagenesis suggest that PokMT1: (i) possesses a large cavity to accommodate the CoA moiety tethered to the substrate, (ii) employs a general acid-base catalysis involving two conserved tyrosine residues, and (iii) uses an unique glutamic acid residue involved in general base catalyzed rearomatization. These findings provide the first insight into the catalytic proficiency, structure, and mechanism of these enzymes, setting the stage to further investigate and exploit the catalytic utility of this unusual class of C-MTs in biocatalysis and synthetic biology.

ASSOCIATE CONTENT Supporting information General materials and methods; accession numbers of the selected C-, N-, and C-MTs used to construct the phylogenetic tree; strains, plasmids and primers used in this study (Table S1, S2 and S2); HR-ESI-MS (Figure S1) and NMR (Figure S2) spectra of 6; SDS-PAGE analysis of purified PokM3 and PokMT1 (Figure S3); size-exclusion chromatography of PokM3 and PokMT1 (Figure S4); in vitro assay of PokMT1 as a C-MT using 4 as an alternative substrate 20


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Biochemistry

(Figure S5); steady-state kinetic analysis of PokM3 and PokMT1 (Figure S6); phylogenetic tree of selected C-, N-, and C-MTs (Figure S7); sequence alignment of the C-MTs that act on aromatic acid substrates (Figure S8); proposed mechanisms for SibL and PokMT1 (Figure S9); the original proposal for the biosynthesis of 1 (Figure S10); and structures of the substrates for the selected CoA ligases and C-MTs used to compare with PokM3 and PokMT1 (Figure S11), respectively. The Supporting Information is available free of charge on the ACS Publication website at DOI:10.1021/acs.biochemistry.xxx.

AUTHOR INFORMATION Corresponding author *E-mail: [email protected]. Telephone: (561) 228-2456. Fax: (561) 228-2472. Author Contributions #

X.G. and I.C. contributed equally to this work.

Acknowledgements This is manuscript #29622 from The Scripps Research Institute. This work is supported in part by the NIH Grants CA078747 and GM115575. I.C. and J.L were supported in part by a German Research Foundation postdoctoral fellowship and a scholarship from China Pharmaceutical University and Chinese Scholarship Council (201607060046), respectively. Notes Conflict of interest The authors declare no conflict of interest.

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Biochemistry

Table 1. Kinetic constants of ligation reaction between CoA and aromatic substrates catalyzed by PokM3 and selected other CoA ligases.

a

enzyme

substrate

b

-1

kcat (s )

Km (M)

kcat/Km

relative

(s-1M-1)

kcat/Kmc

PokM3

4

0.45 ± 0.4

1.3 ± 0.3

0.35

1

MdpB2

4

3.9 x 10-2

4.1 x 10-2

0.95

2.7

benzoate-CoA ligase

Benzoic acid

3.7 x 10-2

0.6–2.0

(6.2–1.8) x 10-2

(17–5.1) x 10-2

phenylacetate-CoA ligase

Phenylacetic acid

40

14

2.9

8.3

4-chlorobenzoate-CoA ligase

4-chlorobenzoic acid

14

1.4

10

29

4-coumaroyl-CoA ligase

4-coumaric acid

0.25

67

3.7 x 10-3

1.1 x 10-2

a

Selected CoA ligases used for comparison: MdpB2,7 Rhodopseudomonas palustris benzoate-CoA ligase,29 Azoarcus evansii

phenylacetate-CoA ligase,30 Alcaligenes sp. AL3007 4-chlorobenzoate-CoA ligase,31,32 and Arabidopsis thaliana 4-coumaroyl-CoA ligase.33 b

See Figure S11A for substrate structures

c

Relative to PokM3

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Page 28 of 30

Table 2. Kinetic constants of methylation reactions catalyzed by PokMT1 and selected other C-MTs as well as the PfoMT O-MT. (AcmI possesses similar kinetic constants to AcmL) enzymea

substrateb

kcat (min-1)

Km (M)

kcat/Km

relative

(min-1 M-1)

kcat/Kmc

PokMT1

5

1.92 ± 0.04

2.2 ± 0.1

0.87

1

SibL

L-3HK

0.080

210

3.8 x 10-4

4.4 x 10-4

D-3HK

0.075

311

2.4 x 10-4

2.8 x 10-4

AcmL

3-HK

0.11

600

1.8 x 10-4

2.1 x 10-4

NovO

DMNB

0.50

26.5

1.8 x 10-2

2.1 x 10-2

CouO

DMMA

2.2

52.0

4.2 x 10-2

4.8 x 10-2

PfoMT

caffeoyl-CoA

-

29.0

-

-

a

Selected C- and O-MTs used for comparison: AcmL,26 NovO,34 CouO,34 and PfoMT8

b

See Figure S11B for substrate structures. Abbreviations: 3HK, 3-hydroxykynurenine; DMNB, desmethyl-novobiocic acid; DMMA,

desmethyl-monoamide. c

Relative to PokMT1

28


Page 29 of 30 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

Biochemistry

Table 3. Measured activity for PokMT1 mutants. PokMT1a

PokMT1 activity in %

wild type

100 ± 12.5

Arg15Ala

2.3 ± 0.3

Tyr140Phe

3.1 ± 0.1

Asn156Ala

33.5 ± 2.9

Glu254Ala

4.3 ± 0.4

Tyr301Phe

20.9 ± 3.3

Tyr140Phe-Tyr301Phe

2.9 ± 0.1

a

See Figure 4 and Figure S9 for the proposed mechanism of PokMT1 with conserved active site residues.

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

Page 30 of 30

Graphic TOC

30


PokMT1 from the Polyketomycin Biosynthetic Machinery of

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