Biochemical synthesis of the medicinal sugar l-gulose using fungal alditol oxidase.

Some rare sugars can be potently medicinal, such as l-gulose. In this study, we present a novel alditol oxidase (fAldOx) from the soil fungus Penicillium sp. KU-1, and its application for the effective production of l-gulose. To the best of our knowledge, this is the first report of a successful direct conversion of d-sorbitol to l-gulose. We further purified it to homogeneity with a ∼108-fold purification and an overall yield of 3.26%, and also determined the effectors of fAldOx. The enzyme possessed broad substrate specificity for alditols such as erythritol (kcat/KM, 355 m-1 s-1), thus implying that the effective production of multiple rare sugars for medicinal applications may be possible.

Biochemical characterization of mismatch-binding protein MutS1 and nicking endonuclease MutL from a euryarchaeon Methanosaeta thermophila.

In eukaryotes and most bacteria, the MutS1/MutL-dependent mismatch repair system (MMR) corrects DNA mismatches that arise as replication errors. MutS1 recognizes mismatched DNA and stimulates the nicking endonuclease activity of MutL to incise mismatch-containing DNA. In archaea, there has been no experimental evidence to support the existence of the MutS1/MutL-dependent MMR. Instead, it was revealed that a large part of archaea possess mismatch-specific endonuclease EndoMS, indicating that the EndoMS-dependent MMR is widely adopted in archaea. However, some archaeal genomes encode MutS1 and MutL homologs, and their molecular functions have not been revealed. In this study, we purified and characterized recombinant MutS1 and the C-terminal endonuclease domain of MutL from a methanogenic archaeon Methanosaeta thermophila (mtMutS1 and the mtMutL CTD, respectively). mtMutS1 bound to mismatched DNAs with a higher affinity than to perfectly-matched and other structured DNAs, which resembles the DNA-binding specificities of eukaryotic and bacterial MutS1 homologs. The mtMutL CTD showed a Mn2+/Ni2+/Co2+-dependent nicking endonuclease activity that introduces single-strand breaks into a circular double-stranded DNA. The nicking endonuclease activity of the mtMutL CTD was impaired by mutagenizing the metal-binding motif that is identical to those of eukaryotic and bacterial MutL endonucleases. These results raise the possibility that not only the EndoMS-dependent MMR but also the traditional MutS1/MutL-dependent MMR exist in archaea.

The GIY-YIG endonuclease domain of Arabidopsis MutS homolog 1 specifically binds to branched DNA structures.

In plant organelle genomes, homeologous recombination between heteroallelic positions of repetitive sequences is increased by dysfunction of the gene encoding MutS homolog 1 (MSH1), a plant organelle-specific homolog of bacterial mismatch-binding protein MutS1. The C-terminal region of plant MSH1 contains the GIY-YIG endonuclease motif. The biochemical characteristics of plant MSH1 have not been investigated; accordingly, the molecular mechanism by which plant MSH1 suppresses homeologous recombination is unknown. Here, we characterized the recombinant GIY-YIG domain of Arabidopsis thaliana MSH1, showing that the domain possesses branched DNA-specific DNA-binding activity. Interestingly, the domain exhibited no endonuclease activity, suggesting that the mismatch-binding domain is required for DNA incision. Based on these results, we propose a possible mechanism for MSH1-dependent suppression of homeologous recombination.

Engineering antimicrobial coating of archaeal poly-γ-glutamate-based materials using non-covalent crosslinkages.

We are now entering a new age of intelligent material development using fine, sustainable polymers from extremophiles. Herein we present an innovative (but simple) means of transforming archaeal poly-γ-glutamate (PGA) into extremely durable polyionic complexes with potent antimicrobial performance. This new supra-polymer material (called PGA/DEQ) was subjected to nuclear magnetic resonance and X-ray diffraction spectroscopies to characterize in structural chemistry. Calorimetric measurements revealed its peculiar thermal properties; to the best of our knowledge, it is one of the most heat-resistant biopolymer-based polyionic complexes developed to date. PGA/DEQ is particularly useful in applications where surface functionalization is important, e.g., antimicrobial coatings. The spontaneously assembled PGA/DEQ coatings (without any additional treatments) were remarkably resistant to certain organic solvents (including chloroform), even at high salt concentrations (theoretically greater than those found in sea water), and various pH values. However, the pH-response tests also implied that the PGA/DEQ coatings could be removed only when concentrated citrate di-salts were used, whereas most crosslinked polymer composites (e.g., thermoset matrices) are difficult to recycle and treat downstream. We also discuss PGA/DEQ-immobilized surfaces that exhibit enigmatic microbicidal mechanisms.

Archaeal MutS5 tightly binds to Holliday junction similarly to eukaryotic MutSγ.

Archaeal DNA recombination mechanism and the related proteins are similar to those in eukaryotes. However, no functional homolog of eukaryotic MutSγ, which recognizes Holliday junction to promote homologous recombination, has been identified in archaea. Hence, the whole molecular mechanism of archaeal homologous recombination has not yet been revealed. In this study, to identify the archaeal functional homolog of MutSγ, we focused on a functionally uncharacterized MutS homolog, MutS5, from a hyperthermophilic archaeon Pyrococcus horikoshii (phMutS5). Archaeal MutS5 has a Walker ATPase motif-containing amino acid sequence that shows similarity to the ATPase domain of MutSγ. It is known that the ATPase domain of MutS homologs is also a dimerization domain. Chemical cross-linking revealed that purified phMutS5 has an ability to dimerize in solution. phMutS5 bound to Holliday junction with a higher affinity than to other branched and linear DNAs, which resembles the DNA-binding specificities of MutSγ and bacterial MutS2, a Holliday junction-resolving MutS homolog. However, phMutS5 has no nuclease activity against branched DNA unlike MutS2. The ATPase activity of phMutS5 was significantly stimulated by the presence of Holliday junction similarly to MutSγ. Furthermore, site-directed mutagenesis revealed that the ATPase activity is dependent on the Walker ATPase motif of the protein. These results suggest that archaeal MutS5 should stabilize the Holliday junction and play a role in homologous recombination, which is analogous to the function of eukaryotic MutSγ.

Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P)+-dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD+ Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD+/NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the ß-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme. IMPORTANCE: In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the following informative results. Several residues in the active site form a hydrophobic cluster, which may be a part of the hydrophobic core essential for protein folding. To our knowledge, there is no previous report demonstrating that a hydrophobic cluster in the active site of any l-amino acid dehydrogenase may have a critical impact on protein folding. Furthermore, our results suggest that this hydrophobic cluster could strictly accommodate l-Trp. These studies show the structural characteristics of l-Trp dehydrogenase and hence would facilitate novel applications of l-Trp dehydrogenase.

Cooperative adsorption of critical metal ions using archaeal poly-γ-glutamate.

Antimony, beryllium, chromium, cobalt (Co), gallium (Ga), germanium, indium (In), lithium, niobium, tantalum, the platinoids, the rare-earth elements (including dysprosium, Dy), and tungsten are generally regarded to be critical (rare) metals, and the ions of some of these metals are stabilized in acidic solutions. We examined the adsorption capacities of three water-soluble functional polymers, namely archaeal poly-γ-glutamate (L-PGA), polyacrylate (PAC), and polyvinyl alcohol (PVA), for six valuable metal ions (Co(2+), Ni(2+), Mn(2+), Ga(3+), In(3+), and Dy(3+)). All three polymers showed apparently little or no capacity for divalent cations, whereas L-PGA and PAC showed the potential to adsorb trivalent cations, implying the beneficial valence-dependent selectivity of anionic polyelectrolytes with multiple carboxylates for metal ions. PVA did not adsorb metal ions, indicating that the crucial role played by carboxyl groups in the adsorption of crucial metal ions cannot be replaced by hydroxyl groups under the conditions. In addition, equilibrium studies using the non-ideal competitive adsorption model indicated that the potential for L-PGA to be used for the removal (or collection) of water-soluble critical metal ions (e.g., Ga(3+), In(3+), and Dy(3+)) was far superior to that of any other industrially-versatile PAC materials.

Poly-γ-glutamate-based Materials for Multiple Infection Prophylaxis Possessing Versatile Coating Performance.

Poly-γ-glutamate (PGA) possesses a nylon-like backbone and polyacrylate-like carboxyl groups, and shows an extraordinary solubility in water. In this study, the effective synthesis and structural analysis of some water-insoluble PGA ion-complexes (PGAICs) using cationic surfactants, hexadecylpyridinium (HDP), dodecylpyridinium, benzalkonium and benzetonium, were examined. We demonstrated their spontaneous coating performance to the surfaces of different materials (i.e., plastics, metals, and ceramics) as potent anti-staphylococcal and anti-Candida agents. The tests against Staphylococcus aureus revealed that, regardless of a variety of materials, PGAICs maintained surface antimicrobial activity, even after the water-soaking treatment, whereas those against Candida albicans indicated that, among PGAICs, PGA/HDP complex is most useful as an anti-fungal agent because of its coating stability. Moreover, the log reduction values against Influenza A and B viruses of PGA/HDP-coated surfaces were estimated to be 5.4 and 3.2, respectively, suggesting that it can be dramatically suppressed the infection of influenza. This is to our knowledge the first observation of PGA-based antiviral coatings.

Extra-chromosomal DNA maintenance in Bacillus subtilis, dependence on flagellation factor FliF and moonlighting mediator EdmS.

Extra-chromosomal DNA maintenance (EDM) as an important process in the propagation and genetic engineering of microbes. Bacillus subtilis EdmS (formerly PgsE), a protein comprising 55 amino acids, is a mediator of the EDM process. In this study, the effect of mutation of global regulators on B. subtilis EDM was examined. Mutation of the swrA gene abolished EdmS-mediated EDM. It is known that swrA predominantly regulates expression of the fla/che operon in B. subtilis. We therefore performed EDM analysis using fla/che-deletion mutants and identified an EDM-mediated EDM cooperator in the flgB-fliL region. Further genetic investigation identified the flagellation factor FliF is a crucial EDM cooperator. To our knowledge, this is the first observation of the moonlighting function of FliF in DNA maintenance.

Rapid purification and plasticization of D-glutamate-containing poly-γ-glutamate from Japanese fermented soybean food natto.

Poly-γ-glutamate (PGA) is a major component of mucilage derived from natto, a Japanese fermented food made from soybeans, and PGAs obtained under laboratory's conditions contain numerous d-glutamyl residues. Natto foods are thus promising as a source for nutritionally safe d-amino acids present in intact and digested polymers, although there is little information on the stereochemistry of PGA isolated directly from natto. Here, we describe the development of a new process for rapid purification of PGA using alum and determined the D-glutamate content of natto PGA by chiral high-performance liquid chromatographic analysis. Further, using hexadecylpyridinium cation (HDP(+)), which is a compound of toothpaste, we chemically transformed natto PGA into a new thermoplastic material, called DL-PGAIC. (1)H nuclear magnetic resonance and calorimetric measurements indicate that DL-PGAIC is a stoichiometric complex of natto PGA and HDP(+) with glass transition points of -16.8 °C and -3.1 °C. Then, DL-PGAIC began decomposing at 210°C, suggesting thermal stability suitable for use as a supramolecular soft plastic.

NRPSs and amide ligases producing homopoly(amino acid)s and homooligo(amino acid)s.

Microorganisms are capable of producing a wide variety of biopolymers. Homopoly(amino acid)s and homooligo(amino acid)s, which are made up of only a single type of amino acid, are relatively rare; in fact, only two homopoly(amino acid)s have been known to occur in nature: poly(ε-L-lysine) (ε-PL) and poly(γ-glutamic acid) (γ-PGA). Bacterial enzymes that produce homooligo(amino acid)s, such as L-ß-lysine-, L-valine-, L-leucine-, L-isoleucine-, L-methionine-, and L-glutamic acid-oligopeptides and poly(α-l-glutamic acid) (α-PGA) have recently been identified, as well as ε-PL synthetase and γ-PGA synthetase. This article reviews the current knowledge about these unique enzymes producing homopoly(amino acid)s and homooligo(amino acid)s.

Microbial production and chemical transformation of poly-γ-glutamate.

Poly-γ-glutamate (PGA), a novel polyamide material with industrial applications, possesses a nylon-like backbone, is structurally similar to polyacrylic acid, is biodegradable and is safe for human consumption. PGA is frequently found in the mucilage of natto, a Japanese traditional fermented food. To date, three different types of PGA, namely a homo polymer of D-glutamate (D-PGA), a homo polymer of L-glutamate (L-PGA), and a random copolymer consisting of D- and L-glutamate (DL-PGA), are known. This review will detail the occurrence and physiology of PGA. The proposed reaction mechanism of PGA synthesis including its localization and the structure of the involved enzyme, PGA synthetase, are described. The occurrence of multiple carboxyl residues in PGA likely plays a role in its relative unsuitability for the development of bio-nylon plastics and thus, establishment of an efficient PGA-reforming strategy is of great importance. Aside from the potential applications of PGA proposed to date, a new technique for chemical transformation of PGA is also discussed. Finally, some techniques for PGA and its derivatives in advanced material technology are presented.

Bacillus subtilis EdmS (formerly PgsE) participates in the maintenance of episomes.

Extrachromosomal DNA maintenance (EDM) is an important process in molecular breeding and for various applications in the construction of genetically engineered microbes. Here we describe a novel Bacillus subtilis gene involved in EDM function called edmS (formerly pgsE). Functional gene regions were identified using molecular genetics techniques. We found that EdmS is a membrane-associated protein that is crucial for EDM. We also determined that EdmS can change a plasmid vector with an unstable replicon and worse-than-random segregation into one with better-than-random segregation, suggesting that the protein functions in the declustering and/or partitioning of episomes. EdmS has two distinct domains: an N-terminal membrane-anchoring domain and a C-terminal assembly accelerator-like structure, and mutational analysis of edmS revealed that both domains are essential for EDM. Further studies using cells of Bacillus megaterium and itsedmS (formerly capE) gene implied that EdmS has potential as a molecular probe for exploring novel EDM systems.

Development of antimicrobial thermoplastic material from archaeal poly-γ-L-glutamate and its nanofabrication.

Here we describe a stoichiometric ion-complex of archaeal poly-γ-L-glutamate (L-PGA) and hexadecylpyridinium cation (HDP(+)), called PGAIC, which shows remarkable chemical resistance and potential as a novel functional thermoplastic. PGAIC films suppressed the proliferation of prokaryotic (Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and Staphylococcus aureus) and eukaryotic (Saccharomyces cerevisiae) microorganisms. Moreover, its antifungal activity was demonstrated against a prevalent species of Candida (Candida albicans) and a filamentous fungus (Aspergillus niger). The minimal inhibitory concentrations were estimated as 0.25 mg mL(-1), and zones of growth inhibition appeared when PGAIC-coated polyethylene terephthalate (PET) films were placed in culture plates, whereas PET had very little effect on fungal growth. Soluble PGAIC thus shows promises as an antimicrobial and as a coating substrate. We also succeeded in synthesizing an L-PGA-based nanofiber using an ethanol solution of PGAIC.

Pivotal Enzyme in Glutamate Metabolism of Poly-g-Glutamate-Producing Microbes.

The extremely halophilic archaeon Natrialba aegyptiaca secretes the L-homo type of poly-g-glutamate (PGA) as an extremolyte. We examined the enzymes involved in glutamate metabolism and verified the presence of L-glutamate dehydrogenases, L-aspartate aminotransferase, and L-glutamate synthase. However, neither glutamate racemase nor D-amino acid aminotransferase activity was detected, suggesting the absence of sources of D-glutamate. In contrast, D-glutamate-rich PGA producers mostly possess such intracellular sources of D-glutamate. The results of our present study indicate that the D-glutamate-anabolic enzyme "glutamate racemase" is pivotal in the biosynthesis of PGA.

Analytical approaches to poly-γ-glutamate: quantification, molecular size determination, and stereochemistry investigation.

Poly-γ-glutamate, a nylon-like polyamide material typically consisting of both enantiomers of glutamate, exhibits reasonable biodegradability and its multi-functionality is attracting particular attention. Thus, its industrial application as a versatile and chiral polymer is in increasing demands. Poly-γ-glutamate is presently synthesized using several biocatalysts (e.g., bacterial cells), but the uncontrollable structural diversity of such biosynthesized materials is an area of concern. This review highlights analytical approaches of interest to assure the functional and structural reproducibility of poly-γ-glutamate.

Moonlighting role of a poly-gamma-glutamate synthetase component from Bacillus subtilis: insight into novel extrachromosomal DNA maintenance.

The Bacillus subtilis structural gene pgsE was investigated as a tool for extrachromosomal DNA maintenance (EDM). It ameliorated the stability of high-copy-number vectors, regardless of whether they were derived from rolling-cycle or theta-mode replicons, without any selective pressure. This unique EDM phenomenon may occur via a trans-acting mechanism.

Bacillus subtilis pgsE (Formerly ywtC) stimulates poly-γ-glutamate production in the presence of zinc.

Poly-γ-glutamate (PGA) is a versatile nylon-like material, and enhanced production of PGA is required for various bio-industrial applications. In this study, we first examined the effects of available sugars on the production of Bacillus subtilis PGA, and demonstrated the good applicability of pentoses (e.g., D-xylose). Then, we characterized the pgsE gene of B. subtilis, which encodes a 6.5-kDa protein of 55 amino acids (PgsE), as a genetic tool for increasing the yield of PGA without changing its structural features (e.g., polymer stereochemistry and molecular size distribution). In the presence of Zn(2+), the induction of PgsE tripled the PGA productivity of B. subtilis subsp. chungkookjang. This finding will contribute to the establishment of an improved PGA-production system.

Extremolyte-like applicability of an archaeal exopolymer, poly-gamma-L-glutamate.

An extremely halophilic archaeon Natrialba aegyptiaca produces extracellular poly-gamma-glutamate (PGA), in which only L-glutamate is polymerized via gamma-amide linkages. We examined the extremolyte-like applicability of archaeal PGA and found the ameliorating effects of L-PGA on the resistibility to freeze-thawing and proteolysis, thermostability, and alkalotolerance of a model enzyme, labile DNA ligase. For example, the coexistence of low (e.g. 0.01 mg mL(-1)) and high (e.g. 0.1 mg mL(-1)) concentrations of L-PGA with an average molecular mass of 1000 kDa increased the midpoint of thermal inactivation of DNA ligase by about 15 degrees C and 18 degrees C, respectively, and the model enzyme further remained active even under extremely alkaline conditions of pH 11.4 in the presence of the high concentration of L-PGA. This is the first characterization of the stereo-regular PGA molecules as atypical extremolytes. L-PGA from extremophiles has great potential as a bio-based protectant (or stabilizer) with industrial versatility.

Identification and biochemical characterization of membranous short-chain polyglutamate from Bacillus subtilis.

It is generally thought that natural strains of Bacillus subtilis produce poly-gamma-glutamate (PGA) as a large exopolymer (over 1,000 kDa) with high water solubility. However, extracellular PGA (ePGA) of B. subtilis is actually diverse in molecular size and configuration. In this study, we identified membranous PGA (mPGA) from both natural and domestic strains of B. subtilis. In contrast to ePGA, mPGA was relatively small and consistently l-glutamate-rich. Genetic analysis revealed that the pgs operon of B. subtilis is responsible for mPGA production as well as ePGA production. Biochemical analyses using the membranous fractions from B. subtilis ssp. chungkookjang indicated that the presence of zinc ions (Zn(2+)) affected both the membrane association of mPGA and in vitro synthesis (elongation) of PGA. Our observations highlighted three important factors that will affect the structural diversity of B. subtilis PGA, namely the occurrence of mPGA, the effects of Zn(2+), and the configuration of glutamate substrate.