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Structural Analysis of the Glycine Oxidase Homolog CmiS2 Reveals a Unique Substrate Recognition Mechanism for Formation of a #-Amino Acid Starter Unit in Cremimycin Biosynthesis Daisuke Kawasaki, Taichi Chisuga, Akimasa Miyanaga, Fumitaka Kudo, and Tadashi Eguchi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00444 • Publication Date (Web): 02 Jun 2019 Downloaded from http://pubs.acs.org on June 3, 2019
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Biochemistry
Structural Analysis of the Glycine Oxidase Homolog CmiS2 Reveals a Unique Substrate Recognition Mechanism for Formation of a βAmino Acid Starter Unit in Cremimycin Biosynthesis Daisuke Kawasaki, Taichi Chisuga, Akimasa Miyanaga,* Fumitaka Kudo and Tadashi Eguchi* Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551, Japan Supporting Information Placeholder
The flavin adenine dinucleotidedependent oxidase CmiS2 catalyzes the oxidation of N-carboxymethyl-3-aminononanoic acid to produce a 3-aminononanoic acid starter unit for biosynthesis of cremimycin, a macrolactam polyketide. Although CmiS2 exhibits sequence similarity with the wellcharacterized glycine oxidase ThiO, the chemical structure of the substrate of CmiS2 is different to that of ThiO substrate glycine. Here, we present biochemical and structural characterization of CmiS2. Kinetic analysis revealed that CmiS2 has a strong preference for N-carboxymethyl-3aminononanoic acid over other substrates such as N-carboxymethyl-3-aminobutanoic acid and glycine, suggesting that CmiS2 recognizes the nonanoic acid moiety of the substrate as well as the glycine moiety. We determined the crystal structure of CmiS2 in complex with a substrate analog; namely, S-carboxymethyl-3-thiononanoic acid, which enabled the identification of key amino acid residues involved in substrate recognition. We discovered that Asn49, Arg243, and Arg334 interact with the carboxyl group of the nonanoic acid moiety, while Pro46, Leu52, and Ile335 recognize the alkyl chain of the nonanoic acid moiety via hydrophobic interaction. These residues are highly conserved in CmiS2 homologs involved in the biosynthesis of related macrolactam polyketides, but are not conserved in glycine oxidases such as ThiO. These results suggest that CmiS2-type enzymes employ a distinct mechanism of substrate recognition for the synthesis of β-amino acids. ABSTRACT:
Cremimycin is a 19-membered macrolactam antibiotic which is produced by Streptomyces sp. MJ635-86F5 and contains a 3-aminononanoic acid (ANA) moiety in its polyketide skeleton.1 ANA is synthesized from non-2-enoyl-acyl carrier protein (ACP) thioester that is constructed via the polyketide pathway (Figure 1).2–4 The thioesterase CmiS1
catalyzes the Michael addition of glycine to the βposition of non-2-enoyl-ACP and subsequent hydrolysis of the thioester to N-carboxymethyl-3aminononanoic acid (CMANA). The flavin adenine dinucleotide (FAD)-dependent oxidase CmiS2 then catalyzes the oxidation of CMANA to produce ANA and glyoxylic acid. Homologs of cmiS1 and cmiS2 are present in the biosynthetic gene clusters for BE14106 (becU and becI), ML-499 (mlaU and mlaI), and heronamide (herU and herI), all of which contain a long-chain β-amino fatty acid within their polyketide backbone (Figure S1).5–7 Therefore, it has been suggested that these β-amino fatty acids are synthesized via the same mechanism. Mechanistic insights into the catalytic reaction and substrate recognition of CmiS1-type enzymes have been elucidated through structural analysis of the CmiS1 homolog SAV606.4,8 However, the substrate recognition mechanism of CmiS2 remains elusive. CmiS2 shows sequence similarity with FADdependent glycine oxidases including ThiO from Bacillus subtilis.9 ThiO is well studied from a mechanistic and structural point of view9–15 and has been suggested to participate in the early stage of thiamine biosynthesis.16 In the proposed reaction mechanism of ThiO, the pro-S hydrogen of the glycine C atom is transferred to FAD as a hydride to form a glyoxyl imine intermediate, which is thought to be used for formation of the thiazole ring prior to hydrolysis.16 It is expected that CmiS2 utilizes a similar reaction mechanism; that is, CmiS2 catalyzes the oxidation of the N-carboxymethyl moiety of CMANA via hydride transfer to FAD, followed by spontaneous hydrolysis to produce ANA and glyoxylic acid (Figure S2). However, it is likely that CmiS2 employs a different substrate recognition mechanism to glycine oxidase, given that the chemical structure of CMANA is considerably different to glycine. In this study, we carried out biochemical and structural analyses of CmiS2 in order to elucidate the substrate recognition mechanism.
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AT
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Figure 1. Illustration of the biosynthetic pathway of 3-aminononanoic acid (ANA) in cremimycin biosynthesis. The ANA unit is shown in red. CmiP4, CmiP3, and CmiP2 represent polyketide synthase (ACP: acyl carrier protein, AT: acyltransferase, DH: dehydratase, ER: enoylreductase, KR: ketoreductase, KS: ketosynthase, KSQ: ketosynthase-like decarboxylase). mM) and an 8-fold lower kcat value (0.165 ± 0.004 sfor racemic CMABA compared with those for CMANA, suggesting that CmiS2 can distinguish alkyl chain length. Furthermore, CmiS2 showed a 14-fold higher Km value (302 ± 29 mM) for sarcosine compared with that for CMABA, and showed no detectable activity against glycine, indicating that the substrate specificity of CmiS2 is different to that of ThiO. These results suggest that CmiS2 recognizes the nonanoic acid moiety of the substrate. We determined the crystal structure of CmiS2 (PDB code 6J38) at 2.30 Å resolution (Figure 2A and Table S2). The CmiS2 molecule consists of two domains, a FAD-binding domain (residues 1–85, 148–222, and 309–368) and a substrate-binding domain (residues 86–147 and 223–308). The CmiS2 molecule contains one noncovalently bound FAD molecule. The adenosine diphosphate-ribosyl moiety is sequestered in the FAD-binding domain, and the reactive isoalloxazine ring is located at the interface between the two domains. The overall structure of CmiS2 is similar to those of ThiO (PDB code 1NG3; root mean square deviation [RMSD], 1.9 Å; sequence identity, 25%) and glycine oxidase18 from Geobacillus kaustophilus (PDB code 4YSH; RMSD, 1.9 Å; sequence identity, 32%). CmiS2 is also structurally similar to sarcosine oxidase19 from Corynebacterium sp. U-96 (PDB code 3AD7, RMSD, 2.5 Å; sequence identity, 21%), D-amino acid oxidase (DAAO)20 from Rhodosporidium toruloides (PDB code 1C0L; RMSD, 2.9 Å; sequence identity, 23%) and the glycine oxidase-like domain (MnmC1) of MnmC21 from Escherichia coli (PDB code 3PS9; RMSD, 2.5 Å; sequence identity, 19%), all of which belong to the FAD-dependent D-amino acid oxidase family.22
The stereochemistry at the β-position of the substrate (CMANA) and product (ANA) in the CmiS2 reaction was unclear, since we used the racemic compound as a substrate for CmiS2 in a previous study.3 Therefore, we first determined the stereochemistry of the product by carrying out a large-scale reaction with CmiS1, CmiS2, glycine, and non-2-enoyl N-acetylcysteamine. After methyl esterification of the enzymatic reaction product, the resulting methyl ester was treated with (S)-αmethoxy-α-(trifluoromethyl)phenylacetyl chloride ((S)-MTPA-Cl), then analyzed by 1H-NMR according to Mosher’s method.17 Analysis of the MTPA derivative of racemic ANA revealed distinguishable signals of the (R)or (S)-ANA-derived diastereomers (Figure S3–S6). The MTPA derivative of (R)-ANA showed signals at 3.69 ppm (attributable to OMe in the methyl nonanoate moiety) and 3.45 ppm (attributable to OMe in MTPA), indicating that the product was an R enantiomer (Figure S6). This suggests that CmiS1 produces (R)-CMANA via Michael addition of glycine and subsequent hydrolysis, and CmiS2 uses this compound as a substrate. This is consistent with a previous report which stated that SAV606, a CmiS1 homolog, produces (R)-N-carboxymethyl-3aminobutanoic acid ((R)-CMABA).8 To investigate the substrate specificity of CmiS2, we carried out kinetic analysis using a horseradish peroxidase-couple assay (Figure S7).10,14 The Km and kcat values for racemic CMANA were 0.169 ± 0.033 mM and 1.40 ± 0.13 s-1, respectively (Table S1). These values are comparable with those of ThiO for glycine (Km = 0.99 mM, kcat = 1.3 s-1) and sarcosine (Km = 0.22 mM, kcat = 1.6 s-1).9 We found CmiS2 to have a 130-fold larger Km value (21.7 ± 1.3
1)
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Biochemistry
Figure 2. A) Overall structures of ligand-free CmiS2 (left) and CmiS2 complexed with S-carboxymethyl-3thiononanoic acid (CMTNA) (right). The FAD-binding and substrate-binding domains are shown in magenta and orange, respectively. Yellow and cyan sticks indicate FAD and CMTNA, respectively. B) The substratebinding pocket of the complex. Substrate-recognition residues are shown as sticks. An Fo–Fc electron density map contoured at 2.5 was constructed before incorporation of the CMTNA molecule. In order to understand the substrate-recognition mechanism, we soaked a CmiS2 crystal in a solution containing CMANA. However, we were unable to observe clear electron density corresponding to CMANA. Therefore, we synthesized a racemic substrate mimic, S-carboxymethyl-3-thiononanoic acid (CMTNA) (see supporting information; Figure S8–S9), which was found to be a competitive inhibitor (Ki = 6.0 ± 0.1 M) (Figure S10). A CmiS2 crystal was soaked in a solution supplemented with CMTNA, which enabled successful determination of the crystal structure of CmiS2 in complex with CMTNA (PDB code 6J39) at 2.45 Å resolution (Figure 2A and Table S2). Electron density corresponding to (R)-CMTNA was clearly observable in the vicinity of the isoalloxazine ring of FAD (Figure 2B). Binding of CMTNA led to a ~2.5 Å movement of the -helix 1 (1; Asn49–Leu52) backbone towards the substrate-binding pocket (Figure S11), although no significant conformational change of the polypeptide backbone was observed in other regions (RMSD of 0.34 Å for the C atom of chain A). In the ligand-free structure, the side-chain of Glu50 is oriented away from the substrate-binding pocket and forms a salt bridge with Arg243; in the CMTNA-bound structure, the side-chain of Glu50 rotates approximately 90º towards the substratebinding pocket and forms a salt bridge with Arg334. The 1 region might function as an active-site lid, switching between open and closed conformations to control substrate binding and product release in a similar manner to other FAD-dependent enzymes such as D-amino acid oxidase.23
The substrate-binding pocket is comprised mainly of nine residues: Pro46, Asn49, Leu52, Arg243, Tyr252, Tyr267, Arg308, Arg334, and Ile335 (Figure 2B). The methylene carbon of the S-carboxymethyl moiety is positioned at a distance of 3.2 Å from the N5 atom of FAD (Figure S12). The carboxyl group of the S-carboxymethyl moiety of CMTNA forms hydrogen bonds with Tyr252 (2.5 Å) and Tyr267 (3.2 Å), and a bidentate salt bridge with Arg308 (2.4 Å and 2.6 Å). The carboxyl group of the nonanoic acid moiety interacts with Arg243 (3.2 Å), Arg334 (2.5 Å), and Asn49 (2.9 Å). In addition, the alkyl chain of the nonanoic acid moiety is positioned in the hydrophobic pocket that is formed by Pro46, Leu52, and Ile335. The presence of this nonanoic acid moiety seems to be important for correct placement of the glycine moiety of the substrate, which could explain the lower activity of CmiS2 towards CMABA and sarcosine (Table S1). Structural comparison with the ThiO–Nacetylglycine complex (PDB code 1NG3) revealed that the S-carboxymethyl moiety of CMTNA occupies almost the same position in the CmiS2 structure as the glycine moiety of N-acetylglycine in the ThiO structure (Figure 3). Tyr252, Arg308, and Arg334 of CmiS2 are conserved in ThiO (Tyr246, Arg302, and Arg329, respectively). Furthermore, Tyr246 and Arg302 of ThiO are located close to the carboxyl group of the glycine moiety of Nacetylglycine. However, the side-chain of Arg329 of ThiO interacts with the carboxyl group of the glycine moiety in ThiO, whereas Arg334 of CmiS2 forms a salt bridge with the carboxyl group of the nonanoic acid moiety. This difference might be due to the
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presence of Tyr267 in CmiS2, which is equivalent to Met261 in ThiO. In CmiS2, Tyr267 interacts with the S-carboxymethyl moiety of CMTNA which prevents Arg334 from accessing the S-carboxymethyl moiety (Figure 3). Residues Pro46, Asn49, Leu52, Arg243, and Ile335 of CmiS2 are not conserved in ThiO (Figure S13), and are responsible for selective recognition of the nonanoic acid moiety by CmiS2. The 1 position of CmiS2 shows a ~5 Å displacement compared with the corresponding 1 of ThiO, which contributes to the relatively expanded substrate-binding pocket of CmiS2 (Figure 3). Movement of 1 enables the alkyl chain of the nonanoic acid moiety to be accommodated in the substrate-binding pocket of CmiS2. Structural comparison with the DAAO–D-alanine complex (PDB code 1C0P) showed that the orientation of Scarboxymethyl moiety of CMTNA is also similar to that of D-alanine in the DAAO structure (Figure S14). Tyr252, Tyr267, and Arg308 of CmiS2 are conserved in DAAO (Tyr223, Tyr238, and Arg285, respectively), although Pro46, Asn49, Leu52, Arg243, Arg334, and Ile335 of CmiS2 are not conserved. Tyr223, Tyr238 and Arg285 similarly interact with the carboxyl group of D-alanine in the DAAO structure.20 However, the position of Tyr223 of DAAO shows a ~2 Å displacement compared with the corresponding Tyr252 of CmiS2. This displacement appears to provide sufficient space to accommodate the methyl group of D-alanine in the substrate-binding pocket of DAAO.
Comparison of the sequence of CmiS2 with those of the CmiS2 homologs BecI, MlaI, and HerI (sequence identity 57%, 54%, and 57%, respectively), as well as with those of glycine oxidases (Figure S13) revealed that Tyr252 and Arg308 of CmiS2 are completely conserved in all enzymes. These residues are important for recognition of the carboxyl group of glycine. Residues involved in recognition of the nonanoic acid moiety (Pro46, Asn49, Leu52, Arg243, and Ile335) are almost conserved among CmiS2 homologs, but not conserved in glycine oxidases. This implies that all CmiS2-type enzymes recognize β-substituted long-chain fatty acids in a similar manner. In conclusion, we have elucidated the structural basis of substrate specificity of the novel FADdependent oxidase CmiS2 in cremimycin biosynthesis. Although CmiS2 shows sequence and structural similarities to glycine oxidases such as ThiO, the substrate specificity of CmiS2 is different to that of glycine oxidases. Furthermore, structural analysis showed that key residues involved in the recognition of fatty acid moieties of β-amino acid substrates are only conserved among this class of macrolactam biosynthetic enzymes. ASSOCIATED CONTENT Supporting Information. Experimental details, Figures S1–S14, and Tables S1–S2 (PDF). Accession Codes Protein Data Bank entries 6J38 and 6J39.
AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected] *E-mail:
[email protected] ORCID
Akimasa Miyanaga: 0000-0003-2219-6051 Fumitaka Kudo: 0000-0002-4788-0063 Tadashi Eguchi: 0000-0002-7830-7104 Author Contributions
AM, FK, and TE designed the research; DK, TC, and AM performed the experiments; DK, TC, AM, FK, and TE analyzed the data; DK, AM, FK, and TE wrote the manuscript. All authors have given approval to the final version of the manuscript.
Figure 3. Structural comparison of the CmiS2 complex (magenta and orange) and ThiO-Nacetylglycine complex (dark gray). FAD molecules in the CmiS2 and ThiO structures are shown as yellow and light gray sticks, respectively. Ligand molecules in the CmiS2 and ThiO structures are shown as cyan and black sticks, respectively.
Funding Sources
This work was supported in part by Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research in Innovative Areas (16H06451 to T.E.), and the Japan Society for the Promotion of Science A3 Foresight Program.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was performed with the approval of the Photon Factory Program Advisory Committee (Proposal 2016G624).
ABBREVIATIONS ACP, acyl carrier protein; ANA, 3-aminononanoic acid; CMABA, N-carboxymethyl-3-aminobutanoic acid; CMANA, N-carboxymethyl-3-aminononanoic acid; CMTNA, S-carboxymethyl-3-thiononanoic acid; DAAO, D-amino acid oxidase; FAD, flavin adenine dinucleotide; MTPA, α-methoxy-α(trifluoromethyl)phenylacetate; RMSD, root mean square deviation.
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