Biochemical and Structural Insights into the Aminotransferase CrmG in

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Biochemical and Structural Insights into the Aminotransferase CrmG in Caerulomycin Biosynthesis Yiguang Zhu, Jinxin Xu, Xiangui Mei, Zhan Feng, Liping Zhang, Qingbo Zhang, Guangtao Zhang, Weiming Zhu, Jinsong Liu, and Changsheng Zhang ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00984 • Publication Date (Web): 29 Dec 2015 Downloaded from http://pubs.acs.org on January 2, 2016

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ACS Chemical Biology

Biochemical and Structural Insights into the Aminotransferase CrmG in Caerulomycin Biosynthesis Yiguang Zhu,†,⊥⊥ Jinxin Xu,‡,⊥⊥ Xiangui Mei,§,⊥⊥ Zhan Feng,‡ Liping Zhang,† Qingbo Zhang,† Guangtao Zhang,† Weiming Zhu,*,§ Jinsong Liu,*,‡ Changsheng Zhang*,†

†

CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key

Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China; ‡

Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health,

Chinese Academy of Sciences, Guangzhou 510530, China; §

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and

Pharmacy, Ocean University of China, Qingdao 266003, China; ⊥

These authors contributed equally.

1

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ABSTRACT Caerulomycin A (CRM A 1) belongs to a family of natural products containing a 2,2'-bipyridyl ring

core

structure,

and

is

currently

under

development

as

a

potent

novel

immunosuppressive agent. Herein, we report the functional characterization, kinetic analysis, substrate specificity, and structure insights of an aminotransferase CrmG in 1 biosynthesis. The aminotransferase CrmG was confirmed to catalyze a key transamination reaction to convert an aldehyde group to an amino group in 1 biosynthetic pathway, preferring L-glutamate and L-glutamine as the amino donor substrates. The crystal structures of CrmG in complex with the cofactor 5'-pyridoxal phosphate (PLP) or 5'-pyridoxamine phosphate (PMP), or the acceptor substrate were determined to adopt a canonical fold-type I of PLP–dependent enzymes with a unique small additional domain. The structure guided site-directed mutagenesis identified key amino acid residues for substrate binding and catalytic activities, thus providing insights into the transamination mechanism of CrmG.

2


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Caerulomycins (CRMs) are members of a small family of natural products containing a 2,2'-bipyridyl ring core structure. Other family members include collismycins,1 pyrisulfoxins,2 streptonigrin,3 and orelline.4 A number of CRMs have been reported from Streptomyces caeruleus and the marine-derived Actinoalloteichus cyanogriseus WH1-2216-6,5-7 and have been shown to exhibit diverse bioactivities ranging from antibacterial to cytotoxic activities.5-7 In particular, CRM A1 (Figure 1) was shown to exhibit novel immunosuppressive function by inducing the generation of regulatory T cells,8 significantly suppressing T cell activation and causing the change in the function of B cells.9 Very recently, 1 was found to exert its immunosuppressive effect by targeting iron in a reversible manner.10 Therefore, 1 was currently under development as an attractive and potent immunosuppressive drug candidate.8-11 The pharmaceutical potential and intriguing structure of 1 have attracted enormous chemical synthesis studies.12-14 Recently, the biosynthetic pathways of 1 and its closely-related analogue collismycin A have been demonstrated to involve a common origin of a hybrid polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) system.15-19 Interestingly, the PKS/NRPS system in 1 was able to synthesize a bipyridyl product CRM L 2 (Figure 1) with an L-leucine attached at C-7,15 and a similar product was also identified in the collismycin A pathway.16 However, the exact mechanism to form the bipyridine core in 1 and collismycin A remains unclear.15-19 Nevertheless, several modification steps in the biosynthesis of 1 and collismycin A have been well established by in vivo characterizing metabolites from gene knockout mutants and in vitro elucidating the 3



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enzyme functions,15-19 which lead to a proposal of the biosynthetic pathway of 1 (Figure 1). In support of this proposal, the functions of an unusual amidohydrolase CrmL to remove the redundant L-leucine (2→3),15 a two-component monooxygenase CrmH to form an oxime functionality (5→6),20 and a methyltransferase CrmM for O-methylation of the hydroxy group at C-4 (6→1),21 have been biochemically demonstrated. However, the enzymatic process of converting 3 to 5, involving the transformation of a carboxylic acid group into a primary amino group (Figure 1), has not been elucidated. It has been speculated that the GirC/GriD-like dehydrogenase components,22 CrmN/CrmO, function to reduce a carboxyl group to an aldehyde group, whereas the conversion of an aldehyde to an amino group may involve the aminotransferase CrmG.15 Aminotransferases are PLP-dependent enzymes that transfer an amino group into a keto group, and are ubiquitous in nature to play important roles in nitrogen metabolism in cells.23 A number of aminotransferases involved in primary metabolism for the biosynthesis of amino acids have been biochemically and structurally well studied to provide detailed mechanistic insights.23-25 Amino group-transferring enzymes have also been found in natural product biosynthetic pathways, usually as tailoring modification enzymes,26 especially those for the biosynthesis of amino sugars,27-29 and 2-deoxystreptamine (2-DOS) in aminoglycosides.30-33 In recent years,

functions

and

crystal

structures

of

several

sugar

nucleotide-dependent

aminotransferases for amino sugar biosynthesis in secondary metabolites have been described.34-39 BtrR, a dual functional aminotransferase for the biosynthesis of 2-DOS in butirosin, has also been biochemically and structurally characterized.40, 4


41

Surprisingly,

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limited examples were available on the biochemical and structural characterization of aminotransferases responsible for scaffold construction/modification in the biosynthesis of bacterial secondary metabolites, including OxyQ (anhydrotetracycline, in vivo studies),42 Neo-18 (neomycin) and BtrB (butirosin),43 PvdH (pyoverdinesiderophore),44 YwfG (anticapsin),45 PctV (pactamycin),46, 47 and GenS2 (gentamycin).48 The crystal structure of PigE,49 an aminotransferase in prodigiosin biosynthesis,50 was recently reported. However, the function of PigE has not been biochemically verified. In this study, we report the functional and structural characterization of CrmG as an aminotransferase to convert an aldehyde into a primary amino group in 1 biosynthesis. Comparison of kinetic parameters of the CrmG reactions toward different amino acceptor substrates indicated that the CrmG-catalyzed transamination should occur before the CrmM-catalyzed O-methylation. The crystal structures of CrmG in complex with cofactors PLP, PMP, or the acceptor substrate CRM M 4 were resolved to adopt a canonical fold-type I of PLP–dependent enzymes with a unique small additional domain. The structure-guided site-directed mutagenesis studies of CrmG have allowed the identification of K344 as the catalytic base for the Schiff base formation and the involvement of several key amino acids (F55, L85, F207, K210, W223, V317, S372 and R486) in cofactor and substrate binding. Selective deletions of the additional domain afforded insoluble CrmG variants, indicating the importance of this unique domain in maintaining the right conformation of CrmG. These cumulative data provided biochemical and structural insights into the transamination

5



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mechanism of CrmG, adding another example of aminotransferases modifying the scaffold of natural products.

RESULTS AND DISCUSSION Inactivation of the crmG Gene. Bioinformatics analysis suggests that the crmG gene encodes

a diaminobutyrate-pyruvate aminotransferase

which is a candidate for

incorporation of the amino group at C-7 in 1.15 To verify this hypothesis, the crmG gene was inactivated by PCR-targeted insertional mutation using previously established method (Figure S1).15, 51 The resulting ∆crmG mutant accumulated two products that are distinct from 1 (Figure 2). One product was characterized to be identical to CRM F 8 by comparing HRESIMS and NMR data (Figure 2, Table S1 and Figure S2) with those of standard 8.6 The other product 7, designated CRM P, was isolated and elucidated as a 4-O-demethyl derivative of 8 by HRESIMS, 1H and 13C NMR spectroscopic data (Table S1 and Figure S3). The molecular formula of 7 was established to be C11H10N2O2 by a HRESIMS peak at m/z 203.0817 [M + H]+ (calcd 203.0815). The comparison of 1H and

13

C NMR data of 7 and 8

revealed that 7 only differed from 8 by the absence of signals of a methoxy group (δH/C 3.92/55.3), that was replaced by an exchangeable hydroxy proton signal (δH 10.69). C-4 in 7 shifted to upfield (-1.7 ppm) while both C-3 (+1.5 ppm) and C-5 (+2.2 ppm) shifted downfield. Thus, the structure of 7 was confirmed.

6


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Preparation of Putative Substrates for CrmG Assays. Interestingly, both products 7 and 8 from the ∆crmG mutant contain a hydroxymethyl group at C-6, but not an aldehyde group as expected. Similarly, hydroxymethyl-containing collismycin analogues were accumulated in the ∆clmAT (encoding a CrmG-like aminotransferase) mutant and were shown as shunt products in collismycin biosynthesis via feeding experiments.17 Thus, we reasoned that the natural substrate of CrmG should contain an aldehyde group as previously proposed (Figure 1).15, 20 Therefore, the compounds CRM M 4 and CRM E 9 were prepared from CRM H 6 and 1 (Figure 3A), respectively, by chemical deoximation.19, 20 The identities of products 4 and 9 were confirmed by comparison of their HRESIMS and NMR spectroscopic data (Table S2; Figure S4 and Figure S5) with those of previously reported 4 and 9.20, 52

Biochemical Characterization of CrmG. For in vitro assays, soluble N-His6-tagged CrmG proteins were produced in E. coli BL21(DE3) harboring the pET28a-based plasmid pCSG2201 (Table S2) and were purified to near-homogeneity by Ni-NTA chromatography (Figure S6). The putative substrates 4 and 9 were subsequently assayed with CrmG in the presence of PLP and L-Gln. CRM M 4 was indeed converted to a product displaying the same retention time (Figure 3B, traces i and ii) and the same molecular mass (m/z 202.0982 [M + H]+, calcd 202.0980) (Figure S7) as those of the standard 5.20 The yield of 5 increased with longer incubation time (Figure S8). The turnover of 4 was not observed in control assays lacking either of CrmG, PLP or L-Gln (Figure 3B, traces iii-v). Also, CrmG was capable of catalyzing the conversion of 9 to 10 (Figure 3B, traces vi and vii). The product 10 7



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was confirmed by HRESIMS (C12H13N3O, m/z 216.1138 [M + H]+, calcd 216.1131) (Figure S9). Therefore, CrmG was unequivocally verified to catalyze the transamination of an aldehyde into an amino group at C-7 during the biosynthesis of 1.

To gain more insights, the steady-state kinetic parameters of CrmG reactions were determined for both substrates 4 and 9 (Figure 3C). CrmG displayed a much higher binding affinity toward 4 (Km 14.51 µM) than 9 (Km 619.46 µM). Comparison of kcat/Km values demonstrated that the catalytic efficiency of CrmG toward 4 (kcat 0.50 min-1, kcat/Km 0.0345 µM-1 min-1) was 23-fold more than that toward 9 (kcat 0.94 min-1, kcat/Km 0.0015 µM-1 min-1), indicating that 4 should be the physiological substrate of CrmG. We have previously shown that the methyltransferase CrmM can methylate 6 to form1, but none of the other tested biosynthetic intermediates, including 2-5 (Figure 1), were observed as CrmM substrates.21 These cumulative data confirm that CrmG-catalyzed transamination should occur before the CrmM-catalyzed methylation. CrmG displayed a slightly lower catalytic efficiency (kcat/Km 0.0345 µM-1 min-1) than those of PctV (kcat/Km 0.186 µM-1 min-1, an aminotransferase in pactamycin biosynthesis),46,

47

and several sugar aminotranferases WecE (kcat/Km 0.207

µM-1 min-1),53 PseC (kcat/Km 0.215 µM-1 min-1),39 and DesI (kcat/Km 0.432 µM-1 min-1).54

It is questionable why 7 and 8, but not the CrmG substrates 4 and 9, were accumulated in the ∆crmG mutant (Figure 2). Given that 4 contains an aldehyde lacking a hydrogen atom in the alpha position, we reason that a Cannizzaro reaction would account for the labile conversion of 4 to 7 under fermentation conditions. Indeed, when the aldehyde 4 was kept at 8


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room temperature for several days in Tris-HCl buffers ranging from pH 6 to pH 8, the aldehyde 4 was spontaneously converted to the carboxylic acid 3 and the alcohol 7 (Figure S10). Similarly, 9 was found to be converted to 8 upon the same treatment (Figure S10). Both 4 and 9 were almost completely converted to 7 and 8, respectively (Figure S10), when stored for 10 days in Tris-HCl buffers of pH 7, similar to fermentation conditions (pH 7) of the ∆crmG mutant (Supporting materials and methods).

Amino Donor Specificity of CrmG. The amino donor specificity of CrmG was probed with various L- and D-amino acids using 4 as an acceptor. Conversions of 4 to 5 was observed for L-Glu, L-Gln, L-Ala, L-Arg, L-Met, L-Orn, and L-Gly (Figure S11), while no activities were detected with other 9L-amino acids, including L-Asn, L-His, L-Ile, L-Leu, L-Lys, L-Phe, L-Pro, L-Ser and L-Thr (Figure S11). It should be noted that CrmG only could recognize L-amino acids, none of the tested D-amino acids (including D-Glu, D-Gln, D-Ala, D-Arg, D-Met and D-Thr) were observed as amino donor substrates for CrmG. When defining the relative enzymatic activity (initial velocity) of CrmG with 4 and L-Gln arbitrarily as 100%, CrmG displayed the best activity with L-Glu (140%) and moderate activity with L-Ala (52%). Only weak activities were observed for L-Arg (26%), L-Orn (25%), L-Met (23%), and L-Gly (5%) (Table S4). For most sugar aminotransferases, the amino donor is either L-Glu or L-Gln, or L-Asp in some cases.28 PctV only recognized L-Glu,46 while YwfG utilized L-Phe.45 Although CrmG preferred L-Glu or L-Gln as amino donors (Table S4), some uncommon amino acids (e.g. L-Ala, L-Arg, L-Orn, L-Met, and L-Gly) also supported CrmG-catalyzed turnover. 9



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Similarly, the aminotransferase Neo-18 in neomycin biosynthesis also exhibited a broad substrate specificity, which preferred L-Glu and L-Gln, but could also accept L-Leu, L-Lys, L-Arg, L-Asp, and S-adenosylmethionine (SAM) as amino donors.43

Crystal Structure of CrmG. To further elucidate the catalytic mechanism, crystal structures of CrmG were determined in PLP, PMP and PMP-4 (the acceptor substrate) bound forms with resolutions of 2.6 Å, 2.46 Å, and 2.3 Å, respectively (Table 1).It should be noted that the PMP-bound crystal of CrmG was obtained in an attempt to co-crystallize CrmG with L-Gln and PLP. However, no co-crystallization of L-Gln was observed. We reason that PMP was generated from PLP by reacting with the amino acid donor L-Gln in the first-half of a typical transaminase reaction.24, 25 All crystals were crystalized in P1 space group and contain two dimers in the asymmetric unit (Figure 4A ,B). The monomer structure of CrmG contains three domains: a large domain, a small domain and a unique additional domain. The large domain and the small domain adopt the fold as (S)-selective ω-transaminases.55, 56 On top of the large domain, CrmG contains a unique additional domain (Figure 4A). This additional domain (residues 139-195 and 255-272) comprises of a two-stranded antiparallel β sheet (β7 and β8) packing against a helix bundle formed by two α helices (C terminal of α4 and α5) and a small helix (η1). This additional domain does not show any structural similarity with known structures in PDB.

Cofactor-Binding Site. An overlay of PLP-bound CrmG structure with its structurally closest homologue, the E. coli N-succinylornithine transaminase AstC monomer,57 reveals a similar 10


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cofactor binding site of CrmG and AstC (Figure 4A). PLP is covalently linked via a Schiff base to the ɛ-amino group of K344 (Figure4C), but the covalent linkage between PMP and K344 is not observed (Figure 4D). A noteworthy structural feature of CrmG when binding with cofactor is that F207 adopts different conformations: F207 packs against the aromatic ring of PMP with a displaced face-to-face stack, whereas F207 forms an edge-to-face pi-pi stack with the aromatic ring of PLP (Figure 4D).

Substrate Recognition. In the crystal structure of CrmG complexed with PMP-4, the clear electron density was observed between C-4' atom of PMP and C-7 of 4 (Figure 5A), indicating the formation of an external aldimine between 4 and PMP. CRM M 4 was trapped in the substrate binding pocket by a number of polar and non-polar interactions (Figure5B). The hydroxy group of 4 interacts with the guanidine group of R486 and the main chain carbonyl of P477 via a water molecule; the nitrogen N1 interacts with S372' from the other monomer via another water molecule. Ring A of 4 (Figure 1) forms face-to-face pi-pi stack with F55; ring B interacts with W223 through edge to face pi-pi stack and interacted with L85' through hydrophobic interaction. Additionally, 4 also forms hydrophobic interactions with F207.

To explore the mechanism of the amino donor selectivity, the intermediates of the first half of the CrmG reaction, PLP-Glu and PLP-Gln, have been docked onto the binding pocket of the PLP-bound CrmG structure, respectively (Figure 5C,D). These modeled CrmG structures highlighted the critical residues interacted both with L-Glu and L-Gln (Figure 11



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5C,D), such as R486 (forming salt bridges with the α-carboxylate), S372' (forming hydrogen bond with the γ-carboxylate), and F55 (hydrophobic interactions). K210 forms a salt bridge with the γ-carboxylate group of L-Glu (Figure 5C). However, L-Gln could not interact with K210 (Figure 5D). This indicates that L-Glu is more stable in the active pocket than L-Gln. As such, L-Glu should be a better amino donor for CrmG than L-Gln, which has been confirmed by biochemical data (Table S4).

Structure-Guided Mutagenesis of CrmG. Guided by the cofactor- and substrate-binding feature of CrmG, several CrmG mutants were made by site-directed mutagenesis. Consistent with the observation that K344 forms an internal Schiff base with PLP (Figure 4C), the K344A mutant completely lost the activity for transamination (Table 2 and Figure S12), confirming that K344 is the essential catalytic base. The V317A mutant also completely lost the transamination activity (Table 2 and Figure S12), implying the important role of V317 in PLP binding (Figure 4C). None of the three mutants F207A, F207L and F207W were able to catalyze the conversion of 4 to 5 (Table 2 and Figure S12), consistent with

that

F207

ω-transaminases.55,

is 57

a

conserved

residue

participating

in

co-factor

binding

of

F55G and W223A mutant displayed reduced activities for

transamination, and the L85D mutant completely demolished the CrmG activity (Table 2 and Figure S12). These observations confirmed the importance of F55, W223, and L85 for 4 recognition as observed in the 4-bound CrmG structures (Figure 5B). R486 interacts with both amino acceptor (4) and amino donor (L-Glu or L-Gln) substrates (Figure 5). 12


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Consequently, the R486E mutant completely demolished the activity of CrmG. S372 also plays an important role in coordinating the binding of substrates (Figure 5). S372F was inactive in biochemical assays while S372A maintained trace activities (Table 2). In the modeled structure, K210 is predicted to form a salt bridge with the γ-carboxylate group of L-Glu (Figure 5). Indeed, K210E mutant completely lost the CrmG activity using L-Glu as an amino donor, although it retained trace activity with L-Gln (Table 2 and Figure S12).

CrmG contains a small additional domain that is not seen in other aminotransferases (Figure 4A). A sequence comparison reveals that this additional domain is likely conserved in CrmG and its highly similar analogues (Figure S13). The precise function of this domain is still unclear. Attempts were made to selectively delete the amino acids involving the formation of the additional domain based on both sequence alignment and structural information (Figure S13). Deletion of the small helix η1 afforded the ∆T166-V172 mutant that kept nearly the same activity as the wild type CrmG (Table 2 and Figure S12). However, no soluble proteins were obtained for the mutants ∆I139-R194 (deleting the helix bundle α4-η1-α5), ∆D180-A195 (deleting α5) and ∆S255-D272 (deleting β7-β8), indicating that the additional domain is important in maintaining the correct conformation of CrmG.

Structural Implications for Catalytic Mechanism. The overall CrmG structures of three forms (Figures 4 and 5) represent three states in CrmG reactions and support the proposed ping-pong mechanism of CrmG catalysis (Figure 6). Like many other aminotransferases in amino acids metabolism,24, 25 the CrmG reaction can be divided into two half reactions. The 13



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first half reaction involves the transfer of the amino group from L-Glu to PLP to form PMP, and the second half reaction deals with the transfer of amino group from PMP to the ketone in CRM M 4 through a reductive amination, in a reverse manner of the first half reaction, to finally release a new amine product CRM N 5 and to regenerate PLP.

Three forms of CrmG structures are almost identical in the active site (Figures 4 and 5). However, an interesting conformation change of the conserved F207 in the active site has been identified. Similar to other ω-transaminases, aromatic ring of F207 was almost perpendicular to aromatic ring of PLP in PLP-bound form (Figure 4C; Figure 7A), we named this conformation of F207 vertical conformation; while, in both PMP and PMP-4 bound form, the aromatic ring of F207 forms displaced face to face stack with aromatic ring of PMP, here we named this conformation of F207 planar conformation (Figure 4D, Figure 7A). In PMP-4 bound form, F207 not only interacts with 4 through hydrophobic interactions, but also form cation-pi interaction with external Schiff base (Figure 7B). We speculate that the planar conformation of F207 plays important roles in stabilizing the external Schiff base and substrate, and further facilitating the external Schiff base formation and transaminases reactions. To support this hypothesis, we calculate the interaction energy between F207 and PLP, between F207 and PMP, and between F207 and PMP-4. As shown in Figure 7C, the calculated potential energy between F207 and PMP in PMP-bound structure is -909 KJ mol-1 while that between F207 and PLP in PLP-bound structure is -1504 KJ mol-1. The potential energy between F207 and PMP-4 in PMP-4 bound structure is -1438 KJ mol-1. These 14


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theoretical calculations suggested that the vertical conformation of F207 is more favorable for co-factor stabilizing, but the planar F207 would facilitate substrate stabilizing, external Schiff base formation during transaminases reactions.

Conclusion By gene inactivation and biochemical assays, we confirmed that CrmG was a dedicated aminotransferase to convert an aldehyde into a primary amine in the biosynthesis of CRM A 1. The occurrence of transamination by CrmG prior to methylation by CrmM (Figure 1), was demonstrated by kinetic parameters of CrmG reactions. The catalytic mechanism of CrmG reaction, similar to other aminotransferases in primary metabolism, was confirmed by three forms of CrmG crystal structures, which represented three states in the CrmG reaction cycle (Figure 6). Although CrmG adopts a canonical fold-type I of PLP–dependent enzymes, the presence of an additional domain in CrmG, and conformational change of F207 during its catalytic cycle, made CrmG unique from other transaminases. This study adds another example of biochemical and structural demonstration of an aminotransferase modifying the scaffold of natural products.

Experimental Section The experimental procedures are available in Supporting Information. ASSOCIATED CONTENT Supporting Information Methods, structural characterization details, and supporting tables and figures as mentioned in the text. This material is available free of charge via the Internet at http://pubs.acs.org. 15



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AUTHORINFORMATION Corresponding Authors *Email: [email protected] (for biochemistry) *Email: [email protected]. (for structure) *Email: [email protected] (for chemistry) Author Contributions §

Y.Z., J.X. and X.M. contributed equally to this work.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS

We thank the staffs at BL19U1 beamline at National Center for Protein Sciences Shanghai (NCPSS) and BL17U beamline at Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, China, for assistance during data collection. We are grateful to the analytical facilities in SCSIO. Financial support was provided by NSFC (31500638, 31470204, 21172204, 31125001, and 31290233), the NSFC-Shandong Joint Fund for Marine Science Research Centers (No. U1406402), the Special Fund for Marine Scientific Research in the Public Interest of China (No. 201405038); the Chinese Academy of Sciences (XDA11030403 and KGZD-EW-606), the Administration of Ocean and Fisheries of Guangdong Province (GD2012-D01-002); and the PhD Start-up Fund of Natural Science Foundation of Guangdong Province, China (2014A030310356).

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caerulomycin and incorporation of picolinic-acid and glycerol into caerulomycin-A. Can. J. Chem. 66, 191-194. (53) Hwang, B. Y., Lee, H. J., Yang, Y. H., Joo, H. S., and Kim, B. G. (2004) Characterization and investigation of substrate specificity of the sugar aminotransferase WecE from E. coli K12. Chem. Biol. 11, 915-925. (54) Zhao, L., Borisova, S., Yeung, S. M., and Liu, H. (2001) Study of C-4 deoxygenation in the biosynthesis of desosamine: evidence implicating a novel mechanism. J. Am. Chem. Soc. 123, 7909-7910. (55) Malik, M. S., Park, E. S., and Shin, J. S. (2012) Features and technical applications of omega-transaminases. Appl. Microbiol. Biotechnol. 94, 1163-1171. (56) Schiroli, D., and Peracchi, A. (2015) A subfamily of PLP-dependent enzymes specialized in handling terminal amines. Biochim. Biophys. Acta 1854, 1200-1211. (57) Newman, J., Seabrook, S., Surjadi, R., Williams, C. C., Lucent, D., Wilding, M., Scott, C., and Peat, T. S. (2013) Determination of the structure of the catabolic N-succinylornithine transaminase (AstC) from Escherichia coli. PLoS One 8, e58298.

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Figure Legends Figure 1. Proposed post-PKS/NPRS biosynthetic pathway for CRM A 1. The CrmG-catalyzed reaction was boxed.

Figure 2. HPLC analysis of metabolite profiles of the ∆crmG mutant (i) and the wild type strain (ii) with UV detection at 313 nm. The chemical structures of 7 and 8 are also shown.

Figure 3. Preparation of substrates and analysis of CrmG-catalyzed reactions. (A) Scheme for preparation of substrate 4 (or 9) from 6 (or 1) through chemical deoximation and CrmG reactions. (B) HPLC analysis of CrmG enzyme assays with UV detection at 313 nm. (i) a CrmGassay for 4; (ii) standard 5; (iii) minus CrmG, (iv) minus PLP, (v) minus L-Gln; (vi) a CrmG assay for 9; (vii) minus CrmG. A typical in vitro CrmG assay was conducted in 50 µL reaction mixture in Tris-Cl buffer (50 mM, pH 8.0), comprising of 200 µM 4 (or 9), 20 µMCrmG, 5 mM L-Gln and 0.5 mM PLP at 28°C for 1 h. (C) Determination of CrmG kinetic parameters for substrates 4 and 9 with 5-200 µM 4 (or 50-5000 µM 9) and 10 mM L-Gln in triplicates.

Figure 4. Overall structure of CrmG and cofactor binding site. (A) The comparison of CrmG monomer to AstC monomer (PDB code 4ADB, colored light pink). Individual domains of CrmG are colored differently, S domain colored limon, L domain colored green, additional domain colored pale green, PLP and covalently-bound K344 of CrmG are shown as stick. (B) Structure of the CrmG dimer. S domains are colored limon and blue, respectively; L domains are colored green and purple blue, respectively; and the additional domains are colored pale green and light blue, respectively. PLP is shown as spheres. (C) Detailed interactions of PLP in PLP-bound form. PLP and covalently-linked K344 are shown in stick; residues critical for PLP binding are shown as stick. (D) Comparison of PLP binding in PLP-bound form and

24


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PMP binding in PMP-bound form. PLP and PLP-bound form CrmG colored green; PMP and PMP-bound form CrmG colored yellow.

Figure 5. Substrate binding pocket of CrmG. (A) Close-up of the 4 binding pocket, PMP-4 is shown as stick, The 2Fo-Fc electron density omit map of the PMP-4 contoured at 1.0 σ is shown as blue mesh. (B) Detailed interactions of 4 in PMP-4 bound form. Critical residues for 4 binding are shown as yellow stick, water molecules involving in 4 binding are shown as red sphere. (C) PLP-Glu docked onto CrmG. PLP-Glu colored violet; the side chain of residues that involve in Glu interaction are shown as yellow stick; PLP and covalently-linked K344 are shown in white stick. (D) PLP-Gln docked onto CrmG. PLP-Gln colored magenta; long dash line represents salt bridge interaction; short dash line represents hydrogen bond.

Figure 6. Proposed catalytic mechanism of CrmG.

Figure 7. F207 responsible for Schiff base and substrate stabilizing. (A) Comparison the conformation of F207 in PLP-bound form (green) with that in PMP (yellow) and PMP-4 bound form (cyan). PLP, PMP, PMP-4 are shown as green, yellow and cyan stick, respectively. (B) In PMP-4 bound form structure, F207 forms cation-pi interaction (green dash) with external Schiff base of PMP-4, and forms hydrophobic interaction with 4. (C) Calculated interaction energy for F207 and PLP in PLP bound form, F207 and PMP in PMP-bound form, F207-(PMP-4) in PMP-4 bound form.

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Table 1. Data Collection and Refinement Statistics for CrmG Structures. PLP

PMP

CRM M 4

P1

P1

Data collection Space group

P1

Cell dimensions a, b, c (Å)

84.08, 84.21, 88.95

α, β, γ (°)

106.71, 109.16, 95.03 a

83.87, 83.86, 88.44

83.79, 83.69, 88.05

106.66, 109.25, 94.84

106.63, 109.09, 95.07

50.51-2.46 (2.51-2.46)

49.25-2.30 (2.34-2.30)

262030 (14943)

398069 (16597)

Resolution (Å)

69.92-2.60 (2.66-2.60)

Total observations

237893 (14234)

Unique reflection

61697 (4204)

75400 (4396)

86965 (4115)

b Rmerge

0.136 (0.547)

0.150 (0.55)

0.144 (0.507)

I/σ

7.4 (2.3)

Completeness (%)

92.8 (89.5)

Redundancy

3.9 (3.4)

Wilson B factor

17.4

6.8 (2.3)

8.1 (2.5)

97.3 (96.3)

92.2 (87.5)

3.5 (3.4)

4.6 (4.0)

24.3

18.7 49.25-2.30

Refinement Resolution (Å)

62.02-2.60

50-2.46

No. reflections

58537

71589

82633

20.65/23.81

19.80/23.93

c

d

Rwork / Rfree (%)

21.13 / 26.11

B-factors Protein

34.4

20.1

27.6

Ligand/ion

32.8

35.6

30.8

19.9

24.6

18.7

Bond lengths (Å)

0.011

0.012

0.014

Bond angles (°)

0.83

0.84

0.70

Most favored (%)

95.37

95.62

96.01

Outlier (%)

0.84

0.39

0.39

PDB No.

5DDS

5DDU

5DDW

Water R.m.s. deviations

e

Ramachandran plotf

a

The values in parentheses refer to statistics in the highest bin. Rmerge=∑hkl∑i|Ii(hkl)- | / ∑hkl∑iIi(hkl), where Ii(hkl) is the intensity of an observation and is the mean value for its unique reflection; Summations are over all reflections. c R-factor =∑h|Fo(h)－Fc(h)|/∑hFo(h), where Fo and Fc are the observed and calculated structure-factor amplitudes, respectively. d R-free was calculated with 5% of the data excluded from the refinement. e Root-mean square-deviation from ideal values. f Categories were defined by MolProbity. b

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Table 2. Relative Enzymatic Activities of CrmG Variants.

CrmG

Amino

% Relative

Mutants

donor

activity

K344A

L-Gln

0

V317A

L-Gln

0

F207A

L-Gln

0

F207L

L-Gln

0

F207W

L-Gln

0

F55G

L-Gln

59

W223A

L-Gln

71

L85D

L-Gln

6

R486E

L-Gln

0

R486E

L-Glu

0

S372F

L-Gln

0

S372A

L-Gln

10

S372A

L-Glu

4

K210E

L-Gln

3

K210E

L-Glu

0

∆T166-V172

L-Gln

98

27



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Figure 1. Proposed post-PKS/NPRS biosynthetic pathway for CRM A 1. The CrmG-catalyzed reaction was boxed.

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Figure 2. HPLC analysis of metabolite profiles of the ∆crmG mutant (i) and the wild type strain (ii) with UV detection at 313 nm. The chemical structures of 7 and 8 are also shown.

29



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Figure 3. Preparation of substrates and analysis of CrmG-catalyzed reactions. (A) Scheme for preparation of substrate 4 (or 9) from 6 (or 1) through chemical deoximation and CrmG reactions. (B) HPLC analysis of CrmG enzyme assays with UV detection at 313 nm. (i) a CrmGassay for 4; (ii) standard 5; (iii) minus CrmG, (iv) minus PLP, (v) minus L-Gln; (vi) a CrmG assay for 9; (vii) minus CrmG. A typical in vitro CrmG assay was conducted in 50 µLreaction mixture in Tris-Cl buffer (50 mM, pH 8.0), comprising of 200 µM 4 (or 9), 20 µMCrmG, 5 mM L-Gln and 0.5 mM PLP at 28°C for 1 h. (C) Determination of CrmG kinetic parameters for substrates 4 and 9 with 5-200 µM 4 (or 50-5000 µM 9) and 10 mM L-Gln in triplicates.

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Figure 4. Overall structure of CrmG and cofactor binding site. (A) The comparison of CrmG monomer to AstC monomer (PDB code 4ADB, colored light pink). Individual domains of CrmG are colored differently, S domain colored limon, L domain colored green, additional domain colored pale green, PLP and covalently-linked K344 of CrmG are shown as stick. (B) Structure of the CrmG dimer. S domains are colored limon and blue, respectively; L domains are colored green and purple blue, respectively; and the additional domains are colored pale green and light blue, respectively. PLP is shown as spheres. (C) Detailed interactions of PLP in PLP-bound form. PLP and covalently-linked K344 are shown in stick; residues critical for PLP binding are shown as stick. (D) Comparison of PLP binding in PLP-bound form and PMP binding in PMP-bound form. PLP and PLP-bound form CrmG colored green; PMP and PMP-bound form CrmG colored yellow.

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Figure 5. Substrate binding pocket of CrmG. (A) Close-up of the 4 binding pocket, PMP-4 is shown as stick, The 2Fo-Fc electron density omit map of the PMP-4 contoured at 1.0 σ is shown as blue mesh. (B) Detailed interactions of 4 in PMP-4 bound form. Critical residues for 4 binding are shown as yellow stick, water molecules involving in 4 binding are shown as red sphere. (C) PLP-Glu docked onto CrmG. PLP-Glu colored violet; the side chain of residues that involve in Glu interactions are shown as yellow stick; PLP and covalently-linked K344 are shown in white stick. (D) PLP-Gln docked onto CrmG. PLP-Gln colored magenta; long dash line represents salt bridge interaction; short dash line represents hydrogen bond.

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COO-

H

Lys344 HOOC

NH2 HN -

O

N

HOOC OP

-

N H Michaelis complex (internal aldimine)

COO- Lys344

O

COO- Lys344 HOOC

NH3

HN -

OP

N H external aldimine

COO- Lys344 HOOC

NH3

O

N

Lys344 HN

-

O

N

N

N

OP

N

HN -

O

HN

NH3 -

OP

H

O

H2N -

OP N H ketimine

N H quinonoid intermediate

Lys344

NH2

H2O N H external aldimine

COO-

Lys344

Lys344

NH2

H2O

OH O

N

O P N H ketimine

COOH OH

NH2

O

N H quinonoid intermediate

CRM N 5

OH

HN -

OP

Figure 6. Proposed catalytic mechanism of CrmG.

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NH2

O

OP

CRM M 4 N H PMP


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Figure 7. F207 responsible for Schiff base and substrate stabilizing. (A) Comparison the conformation of F207 in PLP-bound form (green) with that in PMP (yellow) and PMP-4 bound form (cyan). PLP, PMP, PMP-4 are shown as green, yellow and cyan stick, respectively. (B) In PMP-4 bound form structure, F207 forms cation-pi interaction (green dash) with external Schiff base of PMP-4, and forms hydrophobic interaction with 4. (C) Calculated interaction energy for F207 and PLP in PLP bound form, F207 and PMP in PMP-bound form, F207-(PMP-4) in PMP-4 bound form.

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CrmG-PLP

CrmG-PMP

CrmG-CRM M

OH N N 7

CRM M 4

O

CrmG H

L-glutamate

α-keto-glutarate


Biochemical and Structural Insights into the Aminotransferase CrmG in

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