Identification of a new oligomycin derivative as a specific inhibitor of the alternative peptidoglycan biosynthetic pathway.

Peptidoglycan is an important macromolecule in bacterial cell walls to maintain cell integrity, and its biosynthetic pathway has been well studied. Recently, we demonstrated that some bacteria such as Xanthomonas oryzae, a pathogen causing bacterial blight of rice, used an alternative pathway for peptidoglycan biosynthesis. In this pathway, MurD2, a MurD homolog, catalyzed the attachment of L-Glu to UDP-MurNAc-L-Ala and MurL, which did not show homology to any known protein, catalyzed epimerization of the terminal L-Glu of the MurD2 product to generate UDP-MurNAc-L-Ala-D-Glu. Because the alternative pathway also operates in some other plant pathogens and opportunistic pathogens, specific inhibitors of the alternative pathway could function as pesticides and antibiotics for these pathogens. In this study, we searched for specific inhibitors of the alternative pathway from metabolites produced by actinomycetes and identified a new oligomycin-class polyketide, which was revealed to inhibit the MurD2 reaction, in culture broth of Micromonospora sp. K18-0097.

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.

Optimization of tyrosol-producing pathway with tyrosine decarboxylase and tyramine oxidase in high-tyrosine-producing Escherichia coli.

Tyrosol (4-hydroxyphenylethanol) is a phenolic compound used in the pharmaceutical and chemical industries. However, current supply methods, such as extraction from natural resources and chemical synthesis, have disadvantages from the viewpoint of cost and environmental protection. Here, we developed a tyrosol-producing Escherichia coli cell factory from a high-tyrosine-producing strain by expressing selected tyrosine decarboxylase-, tyramine oxidase (TYO)-, and medium-chain dehydrogenase/reductase (YahK)-encoding genes. The genes were controlled by the strong T7 promoter and integrated into the chromosome because of the advantages over plasmid-based systems. The strain produced a melanin-like pigment as a by-product, which is suggested to be formed from 4-hydroxyphenylacetaldehyde (a TYO product/YahK substrate). By using a culture medium containing a high concentration of glycerol, which was reported to enhance NADH supply required for YahK activity, the final titer of tyrosol reached 2.42 g/L in test tube-scale cultivation with a concomitant decrease in the amount of pigment. These results indicate that chromosomally integrated and T7 promoter-controlled gene expression system in E. coli is useful for high production of heterologous enzymes and might be applied for industrial production of useful compounds including tyrosine and tyrosol.

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.

Structure of lasso peptide epimerase MslH reveals metal-dependent acid/base catalytic mechanism.

The lasso peptide MS-271 is a ribosomally synthesized and post-translationally modified peptide (RiPP) consisting of 21 amino acids with D-tryptophan at the C-terminus, and is derived from the precursor peptide MslA. MslH, encoded in the MS-271 biosynthetic gene cluster (msl), catalyzes the epimerization at the Cα center of the MslA C-terminal Trp21, leading to epi-MslA. The detailed catalytic process, including the catalytic site and cofactors, has remained enigmatic. Herein, based on X-ray crystallographic studies in association with MslA core peptide analogues, we show that MslH is a metallo-dependent peptide epimerase with a calcineurin-like fold. The crystal structure analysis, followed by site-directed mutagenesis, docking simulation, and ICP-MS studies demonstrate that MslH employs acid/base chemistry to facilitate the reversible epimerization of the C-terminal Trp21 of MslA, by utilizing two pairs of His/Asp catalytic residues that are electrostatically tethered to a six-coordination motif with a Ca(II) ion via water molecules.

N-Formimidoylation/-iminoacetylation modification in aminoglycosides requires FAD-dependent and ligand-protein NOS bridge dual chemistry.

Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N-O or O-S accounting for N-formimidoylation or N-iminoacetylation. The resultant product is no longer sensitive to aminoglycoside-modifying enzymes, a strategy that antibiotic-producing species employ to counter drug resistance in competing species.

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.

Ergothioneine production by Corynebacterium glutamicum harboring heterologous biosynthesis pathways.

In this study, Corynebacterium glutamicum was engineered to produce ergothioneine, an amino acid derivative with high antioxidant activity. The ergothioneine biosynthesis genes, egtABCDE, from Mycolicibacterium smegmatis were introduced into wild-type and l-cysteine-producing strains of C. glutamicum to evaluate their ergothioneine production. In the l-cysteine-producing strain, ergothioneine production reached approximately 40 mg L-1 after 2 weeks, and the amount was higher than that in the wild-type strain. As C. glutamicum possesses an ortholog of M. smegmatis egtA, which encodes an enzyme responsible for γ-glutamyl-l-cysteine synthesis, the effect of introducing egtBCDE genes on ergothioneine production in the l-cysteine-producing strain was evaluated, revealing that a further increase to more than 70 mg L-1 was achieved. As EgtBs from Methylobacterium bacteria are reported to use l-cysteine as a sulfur donor in ergothioneine biosynthesis, egtB from Methylobacterium was expressed with M. smegmatis egtDE in the l-cysteine-producing strain. As a result, ergothioneine production was further improved to approximately 100 mg L-1. These results indicate that utilization of the l-cysteine-producing strain and introduction of heterologous biosynthesis pathways from M. smegmatis and Methylobacterium bacteria are effective for improved ergothioneine production by C. glutamicum.

First direct evidence for direct cell-membrane penetrations of polycationic homopoly(amino acid)s produced by bacteria.

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. Although the biological significance of polycationic homopoly(amino acid)s remains unclear, increasing attention has recently been focused on their potential use to achieve cellular internalization. Here, for the first time, we provide direct evidence that two representative bacterial polycationic isopeptides, ε-poly-L-α-lysine (ε-PαL) and ε-oligo-L-ß-lysine (ε-OßL), were internalized into mammalian cells by direct cell-membrane penetration and then diffused throughout the cytosol. In this study, we used clickable ε-PαL and ε-OßL derivatives carrying a C-terminal azide group, which were enzymatically produced and then conjugated with a fluorescent dye to analyze subcellular localization. Interestingly, fluorescent proteins conjugated with the clickable ε-PαL or ε-OßL were also internalized into cells and diffused throughout the cytosol. Notably, a Cre recombinase conjugate with ε-PαL entered cells and mediated the Cre/loxP recombination, and ε-PαL was found to deliver a full-length IgG antibody to the cytosol and nucleus.

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.

Identification of pulvomycin as an inhibitor of the futalosine pathway.

Menaquinone is an essential cofactor in the electron-transfer pathway for bacteria. Menaquinone is biosynthesized from chorismate using either the well-known canonical pathway established by pioneering studies in model microorganisms or the futalosine pathway, which we discovered in Streptomyces. Because Helicobacter pylori, which causes stomach cancer, uses the futalosine pathway and most beneficial intestinal bacteria including lactobacilli use the canonical pathway, the futalosine pathway will be a great target to develop antibiotics specific for H. pylori. Here, we searched for such compounds from metabolites produced by actinomycetes and identified pulvomycin from culture broth of Streptomyces sp. K18-0194 as a specific inhibitor of the futalosine pathway.

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.

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.

High Production of Ergothioneine in Escherichia coli using the Sulfoxide Synthase from Methylobacterium strains.

We previously constructed a heterologous production system for ergothioneine (ERG) in Escherichia coli using five ERG biosynthesis genes (egtABCDE) from Mycobacterium smegmatis. However, significant amounts of hercynine (HER), an intermediate of ERG, as ERG were accumulated, suggesting that the reaction of EgtB catalyzing the attachment of γ-glutamylcysteine (γGC) to HER to yield hercynyl-γ-glutamylcysteine sulfoxide was a bottleneck. In this study, we searched for other EgtBs and found many egtB orthologs in diverse microorganisms. Among these, Methylobacterium strains possessed EgtBs that catalyze the direct conversion of HER into hercynylcysteine sulfoxide with l-cysteine (l-Cys) as a sulfur donor, in a manner similar to those of acidobacterial CthEgtB and fungal Egt1. An in vitro study with recombinant EgtBs from Methylobacterium brachiatum and Methylobacterium pseudosasicola clearly showed that both enzymes accepted l-Cys but not γGC. We reconstituted the ERG production system in E. coli with egtB from M. pseudosasicola; ERG productivity reached 657 mg L-1.

Recent advances in functional analysis of polyunsaturated fatty acid synthases.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid, eicosapentaenoic acid, and arachidonic acid are essential fatty acids for humans. PUFAs are biosynthesized by either desaturases/elongases from oleic acid or PUFA synthases from acetyl units. PUFA synthases are composed of three or four subunits, and each creates a specific PUFA even though the multiple catalytic domains in each subunit are very similar. We recently dissected these PUFA synthases by in vivo and in vitro experiments and elucidated how the enzymes control PUFA profiles. Moreover, for the first time, we converted a practical microalgal docosahexaenoic acid synthase into an eicosapentaenoic acid synthase based on the results.

Off-Loading Mechanism of Products in Polyunsaturated Fatty Acid Synthases.

Marine microorganisms de novo biosynthesize polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid by polyunsaturated fatty acid (PUFA) synthases composed of three or four polypeptides in a manner similar to fatty acid synthases (FASs). FASs usually possess thioesterase (TE) domains to release free fatty acids from acyl carrier protein (ACP)-tethered intermediates. Here, we investigated the off-loading mechanism with microalgal and bacterial PUFA synthases through in vivo and in vitro experiments. The in vitro experiments with acyltransferase (AT)-like domains and acyl-ACP substrates clearly demonstrated that the AT-like domains catalyzed the hydrolysis of acyl-ACPs to yield free fatty acids.

Identification of actinomycin D as a specific inhibitor of the alternative pathway of peptidoglycan biosynthesis.

Peptidoglycan is an indispensable component of bacterial cell walls. We recently discovered an alternative peptidoglycan biosynthetic pathway, which involves two enzymes, MurD2 and MurL, catalyzing the ligation of L-Glu to UDP-MurNAc-L-Ala and epimerization of the terminal L-Glu of the MurD2 product, respectively. Because the pathway operates in Xanthomonas oryze, a pathogen causing bacterial blight of rice, we searched for specific inhibitors from metabolites produced by actinomycetes to obtain a lead compound to function as an agrochemical. Actinomycin D was isolated from Streptomyces parvulus NBRC 13193 as a specific inhibitor of the pathway. In vitro analysis indicated that actinomycin D inhibited the MurD2 reaction.

Identification of the peptide epimerase MslH responsible for d-amino acid introduction at the C-terminus of ribosomal peptides.

A lasso peptide MS-271 is a ribosomally synthesized and post-translationally modified peptide (RiPP) consisting of 21 amino acids with a d-tryptophan (Trp) at its C terminus. The presence of d-amino acids is rare in RiPPs and few mechanisms of d-amino acid introduction have been characterized. Here, we report the identification of MslH, previously annotated as a hypothetical protein, as a novel epimerase involved in the post-translational epimerization of the C-terminal Trp residue of the precursor peptide MslA. MslH is the first epimerase that catalyzes epimerization at the Cα center adjacent to a carboxylic acid in a cofactor-independent manner. We also demonstrate that MslH exhibits broad substrate specificity toward the N-terminal region of the core peptide, showing that MslH-type epimerases offer opportunities in peptide bioengineering.

Subtle Control of Carbon Chain Length in Polyunsaturated Fatty Acid Synthases.

Some marine bacteria synthesize docosahexaenoic acid (DHA; C22) and eicosapentaenoic acid (EPA; C20) by enzyme complexes composed of four subunits (A-D). We recently revealed that ß-ketoacyl synthase (KSC)/chain length factor (CLF)-like domains in the "C" subunit of DHA synthase catalyzed the last elongation step (C20 to C22) even though their amino acid sequences are very similar to those of EPA synthase. To investigate the amino acid residues controlling the product chain length, conserved residues in the KSC/CLF-like domains in DHA synthase were replaced with corresponding EPA synthase residues. Among 12 mutants, two CLF-like domain-mutated genes completely lost DHA productivity and produced trace amounts of EPA when coexpressed with dha-ABD in Escherichia coli, whereas when coexpressed with epa-ABD, they produced the same amounts of EPA as epa-ABCD. These results suggest that the product profiles were subtly controlled by several amino acid residues.