Separation of an ε-poly-L-lysine derivative by solvent extraction under a controlled interfacial potential difference.

This paper describes the availability of a 1,2-dichloroethane (DCE)-water (W) interfacial system under a controlled interfacial potential difference for the separation of polycationic species. The system was applied to the production of polyethylene glycol-modified ε-poly-L-lysine (PEG-εPL). PEG-εPL is produced by a fermentation process, and the crude product contains a significant amount of non-modified εPL, which is hardly separated by conventional chromatographic techniques. Both εPL species exist in fully protonated forms under certain acidic conditions, and an extractant, dibenzo-18-crown-6 (DB18C6), associates with their ammonium groups to stabilize the polycations in DCE. Despite the polydispersity of the samples, the εPL and crude PEG-εPL give well-defined cyclic voltammetric waves due to the DB18C6-assisted transfer of the polycations at the polarizable DB18C6 (DCE) | (W, pH ~ 3) interface with midpoint potentials useful for a rough prediction of ion partition equilibria. Thus, the partition experiment was performed using the DB18C6, Bu4N[(CF3SO2)2N] (DCE) | crude PEG-εPL, Li[(CF3SO2)2N] (W, pH ~ 3) interfacial system, of which the potential difference was controlled to enable selective extraction of polycationic PEG-εPL by partition of the [(CF3SO2)2N]- ion. The extract could be collected from the DCE phase and was found to consist of highly purified PEG-εPL.

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.

Constitutive and high gene expression in the diaminopimelate pathway accelerates ε-poly-L-lysine production in Streptomyces albulus.

Streptomyces albulus NBRC14147 produces a homopoly(amino acid), ε-poly-L-lysine (ε-PL). Due to its antibiotic activity, thermostability, biodegradability, and non-toxicity to humans, ε-PL is used as a food preservative. In this study, homology searches of diaminopimelate (DAP) pathway genes (dapB and dapE), in an S. albulus genome database, were shown to encode predicted enzymes using dapB or dapE in Escherichia coli strain complementation assays. We observed that dapB and dapE transcriptional levels were weak during ε-PL production stages. Therefore, we strengthened this expression using an ermE constitutive promoter. Engineered strains generated faster growth and ε-PL production rates when compared with the control strain. Moreover, maximum ε-PL yields in S. albulus, where dapB was constitutively expressed, were approximately 14% higher when compared with the control strain. These findings showed that enhanced lysine biosynthetic gene expression generated faster and higher ε-PL production levels.

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.

The Assembly-Line Enzymology of Nonribosomal Peptide Biosynthesis.

Peptide natural products constitute a major class of secondary metabolites produced by microorganisms (mostly bacteria and fungi). In the past several decades, researchers have gained extensive knowledge about nonribosomal peptides (NRPs) generated by ribosome-independent systems, namely, NRP synthetases (NRPSs). NRPSs are multifunctional enzymes consisting of semiautonomous domains that form a peptide backbone. Using a thiotemplate mechanism that employs assembly-line logic with multiple modules, NRPSs activate, tether, and modify amino acid building blocks, sequentially elongating the peptide chain before releasing the complete peptide. Adenylation, thiolation, condensation, and thioesterase domains play central roles in these reactions. This chapter focuses on the current understanding of these central domains in NRPS assembly-line enzymology.

Mechanism of S-Adenosyl-l-methionine C-Methylation by Cobalamin-dependent Radical S-Adenosyl-l-methionine Methylase in 1-Amino-2-methylcyclopropanecarboxylic Acid Biosynthesis.

The radical S-adenosyl-l-methionine (SAM) methylase Orf29 catalyzes the C-methylation of SAM in the biosynthesis of 1-amino-2-methylcyclopropanecarboxylic acid. Here, we determined that the methylation product is (4″R)-4″-methyl-SAM. Furthermore, we found that the 5'-deoxyadenosyl radical generated by Orf29 abstracts the pro-R hydrogen atom from the C-4″ position of SAM to generate the radical intermediate, which reacts with methylcobalamin to give (4″R)-4″-methyl-SAM. Consequently, the Orf29-catalyzed C-methylation was confirmed to proceed with retention of configuration.

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.

Bioavailability of Tauropine After Oral Ingestion in Mouse.

In the present study, we investigated the pharmacokinetics of oral ingested tauropine which is a natural taurine derivative found in marine invertebrates, such as abalone, and in mouse. To measure tauropine in the blood, it was derivatized with phenyl isothiocyanate (PITC), and PITC-tauropine was separated by reverse-phase high-performance liquid chromatography (HPLC) and detected by ultraviolet absorbance. Tauropine was detectable in the blood obtained from mice intraperitoneally injected with tauropine. However, it was not detectable in blood obtained from orally treated mice. In conclusion, oral ingested tauropine may be poorly absorbed by the gastrointestinal tract and transported into the blood.

Crystal structure of the adenylation domain from an ε-poly-l-lysine synthetase provides molecular mechanism for substrate specificity.

ε-poly-l-lysine (ε-PL) synthetase (Pls) is a membrane protein that possesses both adenylation and thiolation domains, characteristic of non-ribosomal peptide synthetases (NRPSs). Pls catalyzes the polymerization of l-Lys molecules in a highly specific manner within proteinogenic amino acids. However, this enzyme accepts certain l-Lys analogs which contain small substituent groups at the middle position of the side chain. From the crystal structures of the adenylation domain from NRPSs, the amino acid residues involved in substrate binding can be assumed; however, the precise interactions for better understanding the Pls recognition of l-Lys and its analogs have not yet been fully elucidated. Here, we determined the crystal structure of the adenylation domain of Pls in complex with the intermediate lysyl adenylate at 2.3 Å resolution. This is the first structure determination of the l-Lys activating adenylation domain. The crystal structure reveals that the shape of the substrate-binding pocket determines the specific recognition of l-Lys and its analogs and the electrostatic and hydrogen-bonding interactions further strengthen substrate binding. This study helps us understand the ε-PL synthesis mechanism and contributes to improving our knowledge of the molecular mechanism of NRPS adenylation domains towards their successful application in bioengineering.

Discovery of a Polyamino Acid Antibiotic Solely Comprising l-ß-Lysine by Potential Producer Prioritization-Guided Genome Mining.

While the genome mining approach has enabled the rational exploration of untapped bioactive natural products, in silico identifications of their biosynthetic genes are often unconnected to the actual production of the corresponding molecules in native strains due to the genetic dormancy. We report here the rational discovery of an unexplored cationic homo polyamino acid (CHPA) antibiotic by potential producer prioritization-guided genome mining. Mining the genome of γ-poly-d-diaminobutyric acid (poly-d-Dab)-producing Streptoalloteichus hindustanus NBRC 15115, which was selected based on the finding that the known CHPAs are universally co-produced in pairs, identified a putative CHPA synthetase, PblA, as a potential candidate being expressed actively. Bioinformatic and biochemical analyses of PblA provided the critical clue that its polymer product could be an unusual CHPA consisting of l-ß-lysine. Instrumental analyses of the metabolites from S. hindastanus indeed revealed the production of an unprecedented linear CHPA, ε-poly-l-ß-lysine, concomitantly with poly-d-Dab. The CHPA we discovered exerted excellent antimicrobial activity against a broad spectrum of microorganisms, including bacteria and fungi, and was revealed to show resistance against nonspecific proteolytic enzymes. This study marks the first report of the efficacy of the strain prioritization-guided genome mining strategy for the discovery of bioactive CHPAs.

Molecular and Mechanistic Characterization of PddB, the First PLP-Independent 2,4-Diaminobutyric Acid Racemase Discovered in an Actinobacterial D-Amino Acid Homopolymer Biosynthesis.

We recently disclosed that the biosynthesis of antiviral γ-poly-D-2,4-diaminobutyric acid (poly-D-Dab) in Streptoalloteichus hindustanus involves an unprecedented cofactor independent stereoinversion of Dab catalyzed by PddB, which shows weak homology to diaminopimelate epimerase (DapF). Enzymological properties and mechanistic details of this enzyme, however, had remained to be elucidated. Here, through a series of biochemical characterizations, structural modeling, and site-directed mutageneses, we fully illustrate the first Dab-specific PLP-independent racemase PddB and further provide an insight into its evolution. The activity of the recombinant PddB was shown to be optimal around pH 8.5, and its other fundamental properties resembled those of typical PLP-independent racemases/epimerases. The enzyme catalyzed Dab specific stereoinversion with a calculated equilibrium constant of nearly unity, demonstrating that the reaction catalyzed by PddB is indeed racemization. Its activity was inhibited upon incubation with sulfhydryl reagents, and the site-directed substitution of two putative catalytic Cys residues led to the abolishment of the activity. These observations provided critical evidence that PddB employs the thiolate-thiol pair to catalyze interconversion of Dab isomers. Despite the low levels of sequence similarity, a phylogenetic analysis of PddB indicated its particular relevance to DapF among PLP-independent racemases/epimerases. Secondary structure prediction and 3D structural modeling of PddB revealed its remarkable conformational analogy to DapF, which in turn allowed us to predict amino acid residues potentially responsible for the discrimination of structural difference between diaminopimelate and its specific substrate, Dab. Further, PddB homologs which seemed to be narrowly distributed only in actinobacterial kingdom were constantly encoded adjacent to the putative poly-D-Dab synthetase gene. These observations strongly suggested that PddB could have evolved from the primary metabolic DapF in order to organize the biosynthesis pathway for the particular secondary metabolite, poly-D-Dab. The present study is on the first molecular characterization of PLP-independent Dab racemase and provides insights that could contribute to further discovery of unprecedented PLP-independent racemases.

tRNA-dependent amide bond-forming enzymes in peptide natural product biosynthesis.

In the ribosome-independent biosynthesis of peptide natural products, amino acid building blocks are generally activated in the form of phosphoesters, esters, or thioesters prior to amide bond formation. Following the recent discovery of bacterial enzymes that utilize an aminoacyl ester with a transfer ribonucleic acid (tRNA) in primary metabolism, the number of tRNA-dependent enzymes used in biosynthetic studies of peptide natural products has increased steadily. In this review, we summarize the rapidly growing knowledge base regarding two types of tRNA-dependent enzymes, which are structurally and functionally distinct. Initially, we focus on enzymes with the GCN5-related N-acetyltransferase fold and discuss the catalytic function and aminoacyl-tRNA recognition. Next, newly found peptide-amino acyl tRNA ligases and their ATP-dependent reactions are highlighted.

The Stereocontrolled Biosynthesis of Mirror-Symmetric 2,4-Diaminobutyric Acid Homopolymers Is Critically Governed by Adenylation Activations.

Among the four bioactive cationic homo-poly(amino acids) discovered in nature, two are mirror-image isomers of poly(2,4-diaminobutyric acid) (poly-Dab) whose biosynthesis has long been unexplained. Their structural analogy plausibly suggested that they could share a common biosynthetic pathway utilizing ε-poly(l-lysine) synthetase-like enzymology but with an unprecedented process for enantiomeric inversion of polymer building blocks. To investigate this possibility, we comparatively explored the biosynthesis of poly-l-Dab and its mirror-image isomer poly-d-Dab in Streptomyces celluloflavus USE31 and Streptoalloteichus hindustanus NBRC15115, respectively, through genome mining, genetic inactivation, and heterologous expression combined with biochemical assays. While they shared the same biosynthetic pathway, the poly-d-Dab biosynthetic gene cluster additionally harbored the racemase gene. The critical finding that poly-d-Dab synthetase, in contrast to the synthetase generating the l-isomer, selectively activated d-Dab through adenylation conclusively demonstrated that free diffusible d-Dab preactivationally generated by the racemase is directly activated to be incorporated into the polymer. Our study thus represents the first demonstration of the stereoselective biosynthesis of a nonribosomal peptide governed by adenylation activity for a d-amino acid other than alanine. In silico sequence comparison between poly-Dab synthetases allowed us to identify amino acid residues potentially responsible for the discrimination of Dab enantiomers. Our results will provide significant insight not only for the future discovery of novel bioactive cationic poly(amino acids) but also for the creation of designer nonribosomal peptides with d-configuration.

C-Methylation of S-adenosyl-L-Methionine Occurs Prior to Cyclopropanation in the Biosynthesis of 1-Amino-2-Methylcyclopropanecarboxylic Acid (Norcoronamic Acid) in a Bacterium.

Many pharmacologically important peptides are bacterial or fungal in origin and contain nonproteinogenic amino acid (NPA) building blocks. Recently, it was reported that, in bacteria, a cyclopropane-containing NPA 1-aminocyclopropanecarboxylic acid (ACC) is produced from the L-methionine moiety of S-adenosyl-L-methionine (SAM) by non-canonical ACC-forming enzymes. On the other hand, it has been suggested that a monomethylated ACC analogue, 2-methyl-ACC (MeACC), is derived from L-valine. Therefore, we have investigated the MeACC biosynthesis by identifying a gene cluster containing bacterial MeACC synthase genes. In this gene cluster, we identified two genes, orf29 and orf30, which encode a cobalamin (B12)-dependent radical SAM methyltransferase and a bacterial ACC synthase, respectively, and were found to be involved in the MeACC biosynthesis. In vitro analysis using their recombinant enzymes (rOrf29 and rOrf30) further revealed that the ACC structure of MeACC was derived from the L-methionine moiety of SAM, rather than L-valine. In addition, rOrf29 was found to catalyze the C-methylation of the L-methionine moiety of SAM. The resulting methylated derivative of SAM was then converted into MeACC by rOrf30. Thus, we demonstrate that C-methylation of SAM occurs prior to cyclopropanation in the biosynthesis of a bacterial MeACC (norcoronamic acid).

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.

Enhancement of metabolic flux toward ε-poly-l-lysine biosynthesis by targeted inactivation of concomitant polyene macrolide biosynthesis in Streptomyces albulus.

ε-Poly-l-lysine (ε-PL) produced as a secondary metabolite of Streptomyces albulus has long been used as a natural food preservative in a number of countries, including Japan, the United States, South Korea, and China. To date, numerous studies employing classical biotechnological approaches have been carried out to improve its productivity. Here we report a modern and rational genetic approach to enhancing metabolic flux toward ε-PL biosynthesis. Based on in silico genome analyses, we revealed that S. albulus NBRC14147 produces five antifungal polyene antibiotics-tetramycin A and B, tetrin A and B, and a trace amount of nystatin A1-concomitantly with antimicrobial ε-PL. Targeted inactivation of the biosynthetic gene cluster for tetramycins and tetrins in a nystatin A1 production-deficient mutant completely abolished the production of polyene macrolides, which in turn led to an approximately 20% improvement in ε-PL production that closely correlated with the polyene defects. The biosynthetic flux for ε-PL was thus successfully enhanced by inactivation of the concomitant secondary metabolite biosynthetic pathways. Since this elimination of concomitantly produced metabolites also allows for simpler purification after fermentation production of ε-PL, the rational strain engineering strategy we show here will improve its industrial production.

Moldable Material from ε-Poly-l-lysine and Lignosulfonate: Mechanical and Self-Healing Properties of a Bio-Based Polyelectrolyte Complex.

A moldable material from a natural cationic polyelectrolyte, ε-poly-l-lysine (ε-PL), was prepared by mixing with two lignosulfonates a reagent for research (L-SO3Na) and a commercially available purified lignosulfonate (Pearllex NP). The obtained ε-PL/lignosulfonate complexes demonstrated the ability to be tuned from a rigid form, such as polystyrene or poly(methyl methacrylate), to a soft elastomer form such as silicone by varying the lignosulfonate species and composition. The maximum toughness of the complex (8.4 MJ/m3) was superior to that of ε-PL or lignosulfonate-derived polyelectrolyte complexes. In addition, the ε-PL/lignosulfonate complex showed self-healing properties due to the many reversible ionic bonds in the complex. The preparation process for the novel complex was simple, involving the mixing and drying of an aqueous solution of the polyelectrolyte without any extra reagents (organic solvents, condensation reagents, and cross-linker). Thus, given these many advantages and the excellent biodegradability of the components, the ε-PL/lignosulfonate complex is expected to be useful as a sustainable structural material.

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.

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.

Draft Genome Sequence of the Most Traditional ε-Poly-l-Lysine Producer, Streptomyces albulus NBRC14147.

The genus Streptomyces is known for its secondary metabolite biosynthetic capacities. We report here the draft genome sequence of the most extensively studied ε-poly-l-lysine producer, Streptomyces albulus NBRC14147. Bioinformatic analysis of the 9.6-Mb chromosome identified a large number of secondary metabolite biosynthetic gene clusters.