Peptide Epimerase Responsible for d-Amino Acid Introduction in Poly-γ-glutamic Acid Biosynthesis.

Poly-γ-glutamic acid (PGA) is a natural polymer of d- and/or l-glutamic acid (Glu) linked by isopeptide bonds. We recently showed that PGA synthetase, an enzyme complex composed of PgsB, PgsC, and PgsA, uses only l-Glu for polymerization, and d-Glu residues are introduced by peptide epimerization. However, it remains unclear which of the three enzymes is responsible for epimerization because in vitro functional characterization of the membrane-associated PgsBCA complex has never been successful. Here, we performed gene exchange experiments and showed that PgsA is responsible for the epimerization. Additionally, we identified a region in PgsA that modulates epimerization activity based on homology modeling from the recently solved structure of MslH, which showed 53% identity to PgsA. Our results suggested that d/l-ratios of the PGA product can be altered by introducing amino acid substitutions in this region, which will be useful for the production of PGA with controlled d/l-ratios.

Studies on biosynthetic enzymes leading to structural and functional diversity of microbial natural products.

The primary metabolic pathways, for the most part, have been disclosed in Escherichia coli and Saccharomyces cerevisiae. These pathways were believed to be common among all microorganisms. However, after discovery of an alternative pathway for biosynthesis of isopentenyl diphosphate, the methylerythritol phosphate pathway, genome mining of alternative biosynthetic pathways for primary metabolites has been performed. My collaborators and I focused on the biosynthetic pathways of menaquinone and peptidoglycan because some microorganisms lack ortholog genes of the known biosynthetic pathways for these compounds. I also studied biosynthetic enzymes for secondary metabolites produced by actinomycetes and fungi because they include many unique enzymes. In this review, outlines of these studies are described.

Peptide epimerase-dehydratase complex responsible for biosynthesis of the linaridin class ribosomal peptides.

Grisemycin, salinipeptin, and cypemycin belong to the linaridin class of ribosomally synthesized and posttranslationally modified peptides that contain multiple dehydrobutyrine and D-amino acid residues. The biosynthetic gene clusters of these linaridins lack obvious candidate genes for the dehydratase and epimerase required to introduce dehydrobutyrine and D-amino acid residues, respectively. However, we previously demonstrated that the grisemycin (grm) cluster contained cryptic dehydratase and epimerase genes by heterologous expression of this biosynthetic gene cluster in Streptomyces lividans and proposed that two genes (grmH and grmL) with unknown functions catalyze dehydration and epimerization reactions. In this study, we confirmed that both GrmH and GrmL, which were shown to constitute a protein complex by a co-purification experiment, were required to catalyze the dehydration, epimerization, and proteolytic cleavage of a precursor peptide GrmA by in vivo experiments. Furthermore, we demonstrated that GrmH/GrmL complex accepted salinipeptin and cypemycin precursor peptides, which possess three additional amino acids.

Biosynthetic Gene Cluster of Linaridin Peptides Contains Epimerase Gene.

Salinipeptins belong to the type-A linaridin class of ribosomally synthesized and post-translationally modified peptides (RiPPs) comprising 22 amino acid residues with multiple D-amino acids. Although chirality of other type-A linaridins, such as grisemycin and cypemycin, has not been reported, the biosynthetic gene clusters of type-A linaridins have identical gene organization. Here, we report heterologous expression of grisemycin biosynthetic gene cluster (grm) and show that grisemycin contains multiple D-amino acids, similar to salinipeptins. The heterologous expression experiments also confirm the involvement of a novel peptide epimerase in grisemycin biosynthesis. Gene-deletion experiments indicate that grmL, a single gene with unknown function, is indispensable for grisemycin production. We also show that the presence of D-amino acids is likely a common feature of linaridin natural products by analyzing two other type-A linaridin clusters.

Identification of Cyclopropane Formation in the Biosyntheses of Hormaomycins and Belactosins: Sequential Nitration and Cyclopropanation by Metalloenzymes.

Hormaomycins and belactosins are peptide natural products that contain unusual cyclopropane moieties. Bioinformatics analysis of the corresponding biosynthetic gene clusters showed that two conserved genes, hrmI/belK and hrmJ/belL, were potential candidates for catalyzing cyclopropanation. Using in vivo and in vitro assays, the functions of HrmI/BelK and HrmJ/BelL were established. HrmI and BelK, which are heme oxygenase-like dinuclear iron enzymes, catalyze oxidation of the ϵ-amino group of l-lysine to afford l-6-nitronorleucine. Subsequently, HrmJ and BelL, which are iron- and α-ketoglutarate-dependent oxygenases, effectively convert l-6-nitronorleucine into 3-(trans-2-nitrocyclopropyl)-alanine through C4-C6 bond installation. These observations disclose a novel pathway of cyclopropane ring construction and exemplify the new chemistry involving metalloenzymes in natural product biosynthesis.

Flavonoids from Woodfordia fruticosa as potential SmltD inhibitors in the alternative biosynthetic pathway of peptidoglycan.

SmltD is an ATP-dependent ligase that catalyzes the condensation of UDP-MurNAc-l-Ala and l-Glu to form UDP-MurNAc-l-Ala-l-Glu, in the newly discovered peptidoglycan biosynthesis pathway of a Gram-negative multiple-drug-resistant pathogen, Stenotrophomonas maltophilia. Phytochemical investigation of the 70% ethanol extract from Woodfordia fruticosa flowers collected in Myanmar led to the identification of anti-SmltD active flavonoids, kaempferol 3-O-(6''-galloyl)-ß-d-glucopyranoside (1), astragalin (2), and juglalin (3). Among them, 1 showed the most potent SmltD inhibitory activity. An enzyme steady-state kinetic study revealed that 1 exerted competitive inhibition with respect to ATP. The results of this study provided an attractive foundation for the further development of novel inhibitors of SmltD.

Discovery of an alternative pathway of peptidoglycan biosynthesis: A new target for pathway specific inhibitors.

Peptidoglycan in bacterial cell walls is a biopolymer consisting of sugars and amino acids and plays important role in maintaining cell integrity from the environment. Its biosynthesis is a major target for antibiotics and the genes and enzymes involved in the biosynthetic pathway have been well studied. However, we recently identified an alternative pathway in the early stage of peptidoglycan biosynthesis in Xanthomonas oryzae, a plant pathogen causing bacterial blight disease of rice. The distribution of the alternative pathway is limited to relatively few bacterial genera that contain many pathogenic species, including Xylella and Stenotrophomonas, besides Xanthomonas. Thus, the alternative pathway is an attractive target for the development of narrow-spectrum antibiotics specific to pathogens. In this minireview, we summarize the discovery of the alternative pathway and identification of its specific inhibitors.

In vitro characterization of MitE and MitB: Formation of N-acetylglucosaminyl-3-amino-5-hydroxybenzoyl-MmcB as a key intermediate in the biosynthesis of antitumor antibiotic mitomycins.

Mitomycins, produced by several Streptomyces strains, are potent anticancer antibiotics that comprise an aziridine ring fused to a tricyclic mitosane core. Mitomycins have remarkable ability to crosslink DNA with high efficiency. Despite long clinical history of mitomycin C, the biosynthesis of mitomycins, especially mitosane core formation, remains unknown. Here, we report in vitro characterization of three proteins, MmcB (acyl carrier protein), MitE (acyl AMP ligase), and MitB (glycosyltransferase) involved in mitosane core formation. We show that 3-amino-5-hydroxybenzoic acid (AHBA) is first loaded onto MmcB by MitE at the expense of ATP. MitB then catalyzes glycosylation of AHBA-MmcB with uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) to generate a key intermediate, GlcNAc-AHBA-MmcB, which contains all carbon and nitrogen atoms of the mitosane core. These results provide important insight into mitomycin biosynthesis.

Ergothioneine production with Aspergillus oryzae.

To establish a reliable and practical ergothioneine (ERG) supply, we employed fermentative ERG production using Aspergillus oryzae, a fungus used for food production. We heterologously overexpressed the egt-1 and -2 genes of Neurospora crassa in A. oryzae and succeeded in producing ERG (231.0 mg/kg of media, which was 20 times higher than the wild type). Abbreviations: ERG: ergothioneine; HER: hercynine; Cys-HER: hercynylcysteine-sulfoxide; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine; l-His: l-histidine; l-Cys: l-cysteine; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry.

Searching for potent and specific antibiotics against pathogenic Helicobacter and Campylobacter strains.

Menaquinone is an obligatory component of the electron-transfer pathway in microorganisms. Its biosynthetic pathway was established by pioneering studies with Escherichia coli and it was revealed to be derived from chorismate by Men enzymes. However, we identified an alternative pathway, the futalosine pathway, operating in some microorganisms including Helicobacter pylori and Campylobacter jejuni, which cause gastric carcinoma and diarrhea, respectively. Because some useful intestinal bacteria, such as lactobacilli, use the canonical pathway, the futalosine pathway is an attractive target for development of chemotherapeutics for the abovementioned pathogens. In this mini-review, we summarize compounds that inhibit Mqn enzymes involved in the futalosine pathway discovered to date.

Control Mechanism for cis Double-Bond Formation by Polyunsaturated Fatty-Acid Synthases.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans. Some microorganisms biosynthesize these PUFAs through PUFA synthases composed of four subunits with multiple catalytic domains. These PUFA synthases each create a specific PUFA without undesirable byproducts, even though the multiple catalytic domains in each large subunit are very similar. However, the detailed biosynthetic pathways and mechanisms for controlling final-product profiles are still obscure. In this study, the FabA-type dehydratase domain (DHFabA ) in the C-subunit and the polyketide synthase-type dehydratase domain (DHPKS ) in the B-subunit of ARA synthase were revealed to be essential for ARA biosynthesis by in vivo gene exchange assays. Furthermore, in vitro analysis with truncated recombinant enzymes and C4 - to C8 -acyl ACP substrates showed that ARA and EPA synthases utilized two types of DH domains, DHPKS and DHFabA , depending on the carbon-chain length, to introduce either saturation or cis double bonds to growing acyl chains.

Control Mechanism for Carbon-Chain Length in Polyunsaturated Fatty-Acid Synthases.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are essential fatty acids. PUFA synthases are composed of three to four subunits and each create a specific PUFA without undesirable byproducts. However, detailed biosynthetic mechanisms for controlling final product profiles have been obscure. Here, the bacterial DHA and EPA synthases were carefully dissected by in vivo and in vitro experiments. In vitro analysis with two KS domains (KSA and KSC ) and acyl-acyl carrier protein (ACP) substrates showed that KSA accepted short- to medium-chain substrates while KSC accepted medium- to long-chain substrates. Unexpectedly, condensation from C18 to C20 , the last elongation step in EPA biosynthesis, was catalyzed by KSA domains in both EPA and DHA synthases. Conversely, condensation from C20 to C22 , the last elongation step for DHA biosynthesis, was catalyzed by the KSC domain in DHA synthase. KSC domains therefore determine the chain lengths.

Biosynthetic Gene Cluster of a d-Tryptophan-Containing Lasso Peptide, MS-271.

MS-271, produced by Streptomyces sp. M-271, is a lasso peptide natural product comprising 21 amino acid residues with a d-tryptophan at its C terminus. Because lasso peptides are ribosomal peptides, the biosynthesis of MS-271, especially the mechanism of d-Trp introduction, is of great interest. The MS-271 biosynthetic gene cluster was identified by draft genome sequencing of the MS-271 producer, and it was revealed that the precursor peptide contains all 21 amino acid residues including the C-terminal tryptophan. This suggested that the d-Trp residue is introduced by epimerization. Genes for modification enzymes such as a macrolactam synthetase (mslC), precursor peptide recognition element (mslB1), cysteine protease (mslB2), disulfide oxidoreductases (mslE, mslF), and a protein of unknown function (mslH) were found in the flanking region of the precursor peptide gene. Although obvious epimerase genes were absent in the cluster, heterologous expression of the putative MS-271 cluster in Streptomyces lividans showed that it contains all the necessary genes for MS-271 production including a gene for a new peptide epimerase. Furthermore, a gene-deletion experiment indicated that MslB1, -B2, -C and -H were indispensable for MS-271 production and that some interactions of the biosynthetic enzymes were essential for the biosynthesis of MS-271.

Enzymatic Formation of a Skipped Methyl-Substituted Octaprenyl Side Chain of Longestin (KS-505a): Involvement of Homo-IPP as a Common Extender Unit.

Longestin (KS-505a), a specific inhibitor of phosphodiesterase, is a meroterpenoid that consists of a unique octacyclic terpene skeleton with branched methyl groups at unusual positions (C1 and C12). Biochemical analysis of Lon23, a methyltransferase involved in the biosynthesis of longestin, demonstrated that it methylates homoisopentenyl diphosphate (homo-IPP) to afford (3Z)-3-methyl IPP. This compound, along with IPP, is selectively accepted as extender units by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homologue, to yield dimethylated GGPP (dmGGPP). The absolute configuration of dmGGPP was determined to be (4R,12R) by degradation and chiral GC analysis. These findings allowed us to propose an enzymatic sequence for key steps of the biosynthetic pathway of the unusual homoterpenoid longestin.

A Glycopeptidyl-Glutamate Epimerase for Bacterial Peptidoglycan Biosynthesis.

d-Glutamate (Glu) supplied by Glu racemases or d-amino acid transaminase is utilized for peptidoglycan biosynthesis in microorganisms. Comparative genomics has shown that some microorganisms, including Xanthomonas oryzae, perhaps have no orthologues of these genes. We performed shotgun cloning experiments with a d-Glu auxotrophic Escherichia coli mutant as the host and X. oryzae as the DNA donor. We obtained complementary genes, XOO_1319 and XOO_1320, which are annotated as a hypothetical protein and MurD (UDP-MurNAc-l-Ala-d-Glu synthetase), respectively. By detailed in vitro analysis, we revealed that XOO_1320 is an enzyme to ligate l-Glu to UDP-MurNAc-l-Ala, providing the first example of MurD utilizing l-Glu, and that XOO_1319 is a novel enzyme catalyzing epimerization of the terminal l-Glu of the product in the presence of ATP and Mg2+. We investigated the occurrence of XOO_1319 orthologues and found that it exists in some categories of microorganisms, including pathogenic ones.

Biosynthesis of Oligopeptides Using ATP-Grasp Enzymes.

Peptides are biologically occurring oligomers of amino acids linked by amide bonds and are indispensable for all living organisms. Many bioactive peptides are used as antibiotics, antivirus agents, insecticides, pheromones, and food preservatives. Nature employs several different strategies to form amide bonds. ATP-grasp enzymes that catalyze amide bond formation (ATP-dependent carboxylate-amine ligases) utilize a strategy of activating carboxylic acid as an acylphosphate intermediate to form amide bonds and are involved in many different biological processes in both primary and secondary metabolisms. The recent discovery of several new ATP-dependent carboxylate-amine ligases has expanded the diversity of this group of enzymes and showed their usefulness for generating oligopeptides. In this review, an overview of findings on amide bond formation catalyzed by ATP-grasp enzymes in the past decade is presented.

A peptide ligase and the ribosome cooperate to synthesize the peptide pheganomycin.

Peptide antibiotics are typically biosynthesized by one of two distinct machineries in a ribosome-dependent or ribosome-independent manner. Pheganomycin (PGM (1)) and related analogs consist of the nonproteinogenic amino acid (S)-2-(3,5-dihydroxy-4-hydroxymethyl)phenyl-2-guanidinoacetic acid (2) and a proteinogenic core peptide, making their origin uncertain. We report the identification of the biosynthetic gene cluster from Streptomyces cirratus responsible for PGM production. Unexpectedly, the cluster contains a gene encoding multiple precursor peptides along with several genes plausibly encoding enzymes for the synthesis of amino acid 2. We identified PGM1, which has an ATP-grasp domain, as potentially capable of linking the precursor peptides with 2, and validate this hypothesis using deletion mutants and in vitro reconstitution. We document PGM1's substrate permissivity, which could be rationalized by a large binding pocket as confirmed via structural and mutagenesis experiments. This is to our knowledge the first example of cooperative peptide synthesis achieved by ribosomes and peptide ligases using a peptide nucleophile.

Biosynthesis of the Carbonylmethylene Structure Found in the Ketomemicin Class of Pseudotripeptides.

We recently discovered novel pseudotripeptides, the ketomemicins, which possess a C-terminal pseudodipeptide connected with a carbonylmethylene instead of an amide bond, through heterologous expression of gene clusters identified in actinobacteria. The carbonylmethylene structure is a stable isostere of the amide bond and its biological significance has been shown in several natural and synthetic products. Despite the biological importance of these compounds, little is known about how the carbonylmethylene structure is biosynthesized. In this work, we fully characterized the biosynthetic machinery of the pseudodipeptide. An aldolase, dehydratase, PLP-dependent glycine-C-acetyltransferase, and dehydrogenase were involved in the formation of the pseudodipeptide, with malonyl-CoA and phenylpyruvate as starter substrates.

Synthesis of Acylborons by Ozonolysis of Alkenylboronates: Preparation of an Enantioenriched Amino Acid Acylboronate.

A concise synthesis of acylborons was achieved by ozonolysis of alkenyl MIDA (N-methyliminodiacetic acid) boronates. This reaction exhibits excellent functional-group tolerance and is applicable to various acyl MIDA boronates and potassium acyltrifluroborates (KATs) which could not be synthesized by previous methods. In addition, α-amino acylborons, which would be essential for peptide ligations, were prepared for the first time. The acylboron of l-alanine was obtained in high enantiopurity and found to be configurationally stable. Oligopeptide synthesis between the α-amino KATs and amino acid in dilute aqueous media was studied.

Characterization of three amidinotransferases involved in the biosynthesis of ketomemicins.

We recently reported a novel class of amide bond forming enzymes (peptide ligases) involved in the biosynthesis of pheganomycins, resorcinomycins and ketomemicins. This class of enzymes exclusively utilizes Nα-amidino amino acids as the N-terminal substrate. In this Letter, we characterized three new amidinotransferases involved in the biosynthesis of ketomemicins and showed that l-arginine was the amidino-acceptor of amidinotransferases in both the Micromonospora sp. and Streptomyces mobaraensis clusters, while the Salinispora tropica enzyme recognized l-valine. Unexpectedly, the S. tropica enzyme accepted several different amino acids as amidino acceptors in addition to l-valine. Accordingly, we re-investigated the specific metabolites governed by the gene cluster of S. tropica and identified several minor congeners of ketomemicin C with different N-terminal amidino-amino acids. These results indicate that the amidinotransferase of S. tropica is promiscuous and could be useful to generate new ketomemicin-type natural products.