Masato Otagiri

Complex Formation by the mrpABCDEFG Gene Products, Which Constitute a Principal Na+/H+ Antiporter in Bacillus subtilis

The Bacillus subtilis Mrp (also referred to as Sha) is a particularly unusual Na(+)/H(+) antiporter encoded by mrpABCDEFG. Using His tagging of Mrp proteins, we showed complex formation by the mrpABCDEFG gene products by pull-down and blue native polyacrylamide gel electrophoresis analyses. This is the first molecular evidence that the Mrp is a multicomponent antiporter in the cation-proton antiporter 3 family.

Cloning, expression and nucleotide sequence of the l-2,3-butanediol dehydrogenase gene from Brevibacterium saccharolyticum C-1012

Abstract A 3-kbp DNA fragment including the l -2,3-butanediol dehydrogenase ( l -BDH) gene ( bud C) from the chromosomal DNA of Brevibacterium saccharolyticum C-1012 was cloned in Escherichia coli JM109 after its insertion into pBluescript II SK + , and the resulting plasmid was named pLBD-SK. The bud C had an open reading frame consisting of 774 bp and encoded 258 amino acids. It was not included in a 2,3-butanediol operon such as is seen in the case of the meso -BDH gene ( bud C) of Klebsiella pneumoniae . For the expression of the bud C, the deletion plasmid pLBD2-119 was prepared from pLBD-SK. E. coli JM109/pLBD2-119 had higher l -BDH activity than that of Br. saccharolyticum C-1012. The l -BDH appeared as two bands on disc-PAGE. Isopropyl-β- d -thiogalacto-pyranoside (IPTG) influenced the quantity radio of the electrophoretic isoenzymes of l -BDH from E. coli JM109/pLBD2-119 that is, a higher relative mobility band with weak substrate specificity was abundantly produced by IPTG. The BDH was considered to belong to the short-chain dehydrogenase/reductase (SDR) family on the basis of the following distinctive features: it possessed two conservative sequences GXXXGXG and YXXXK, and it consisted of about 250 amino acids. As a result of a phylogenetic analysis of SDR family enzymes, the BDHs were considered to comprise a cluster independent from the other SDR enzymes.

Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases

2,3‐Butanediol dehydrogenase (BDH) catalyzes the NAD‐dependent redox reaction between acetoin and 2,3‐butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l‐BDH with a bound inhibitor at 2.0 Å. Comparison with the inhibitor binding mode of meso‐BDH highlights the role of a hydrogen‐bond from a conserved Trp residue192. Site‐directed mutagenesis of three active site residues of meso‐BDH, including Trp190, which corresponds to Trp192 of l‐BDH, converted its stereospecificity to that of l‐BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.

High-throughput recombinant gene expression systems in Pichia pastoris using newly developed plasmid vectors.

We describe here the construction of Gateway-compatible vectors, pBGP1-DEST and pPICZα-DEST, for rapid and convenient preparation of expression plasmids for production of secretory proteins in Pichia pastoris. Both vectors direct the synthesis of fusion proteins consisting of the N-terminal signal and pro-sequences of Saccharomyces cerevisiae α-factor, the recognition sites for Kex2 and Ste13 processing proteases, the mature region of a foreign protein flanked by attB1- and attB2-derived sequences at N- and C-termini, respectively, and myc plus hexahistidine tags added at the extreme C-terminus. To test the usefulness of these vectors, production of endo-glucanases and xylanases from termite symbionts, as well as a fungal glucuronoyl esterase, was performed. Enzyme activities were detected in the culture supernatants, indicating that the chimeric proteins were synthesized and secreted as designed.

Structural and Biochemical Analyses of Glycoside Hydrolase Family 26 β-Mannanase from a Symbiotic Protist of the Termite Reticulitermes speratus

Background: Symbiotic protists of the termite gut contribute to lignocellulosic biomass degradation. Results: A novel protistan β-mannanase efficiently degrades glucomannan and displays glucose/mannose binding properties when complexed with gluco-manno-oligosaccharide. Conclusion: Specific recognition and accommodation of glucose at the distal - subsites provides the structural basis for activity against glucomannan. Significance: The mechanism underlying heteropolysaccharide recognition by mannanase has been clarified. Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of β-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. Here, we report biochemical and structural analyses of glycoside hydrolase family 26 mannanase C (RsMan26C) from a symbiotic protist of the termite Reticulitermes speratus. RsMan26C was characterized based on its catalytic efficiency toward glucomannan, compared with pure mannan. The crystal structure of RsMan26C complexed with gluco-manno-oligosaccharide(s) explained its specificities for glucose and mannose at subsites −5 and −2, respectively, in addition to accommodation of both glucose and mannose at subsites −3 and −4. RsMan26C has a long open cleft with a hydrophobic platform of Trp94 at subsite −5, facilitating enzyme binding to polysaccharides. Notably, a unique oxidized Met85 specifically interacts with the equatorial O-2 of glucose at subsite −3. Our results collectively indicate that specific recognition and accommodation of glucose at the distal negative subsites confers efficient degradation of the heteropolysaccharide by mannanase.

The GH26 β-mannanase RsMan26H from a symbiotic protist of the termite Reticulitermes speratus is an endo-processive mannobiohydrolase: heterologous expression and characterization.

Symbiotic protists in the gut of termites are prominent natural resources for enzymes involved in lignocellulose degradation. Here we report expression, purification, and biochemical characterization of a glycoside hydrolase family 26 mannanase RsMan26H from the symbiotic protist of the lower termite, Reticulitermes speratus. Biochemical analysis of RsMan26H demonstrates that this enzyme is an endo-processive mannobiohydrolase producing mannobiose from oligo- and polysaccharides, followed by a minor accumulation of oligosaccharides larger than mannobiose. To our knowledge, this is the first report describing the unique mannobiohydrolase enzyme from the eukaryotic origin.

Functional Expression of Three Rieske Non-Heme Iron Oxygenases Derived from Actinomycetes in Rhodococcus Species for Investigation of Their Degradation Capabilities of Dibenzofuran and Chlorinated Dioxins

The activity of Rieske non-heme iron oxygenases (aromatic hydrocarbon dioxygenases, AhDOs) is important for the bacterial degradation of aromatic pollutants such as polycyclic aromatic hydrocarbons and dioxins. During our analysis of the role of AhDOs in dioxin bioremediation, some enzymes derived from high G + C Gram-positive actinomycetes were difficult to produce in active form in the Escherichia coli protein expression system. In this study, we constructed a heterologous expression system for AhDOs in Rhodococcus species using a constitutive expression promoter, P dfdB, and a shuttle vector, pRK401, and analyzed the ability of these enzymes to degrade dibenzofuran and deplete several chlorinated dioxins. Three active AhDOs expressed in Rhodococcus strains that were difficult to obtain by the E. coli system showed different regiospecificities for dibenzofuran bioconversion as well as different substrate depletion specificities for chlorinated dioxins. Moreover, AhDO derived from R. erythropolis TA421 showed relatively diverse depletion-substrate specificity for chlorinated dioxins.

Crystallization And Preliminary X-Ray Studies Of L-(+)-2,3-Butanediol Dehydrogenase From Brevibacterium Saccharolyticum C-1012

L(+)-2,3 -Butanediol dehydrogenase (L-BDH) from Brevibacterium saccharolyticum C-1012 has been crystallized by the hanging drop vapor diffusion method with polyethylene glycol 4000 as the precipitant. Crystals of L-BDH belong to the triclinic system, space group P1 with cell dimensions a = 60.8 A, b = 69.2 A, c = 127.4 A , alpha = 96.1 , beta = 100.2 and gama=109.6 . Crystals diffracted to 2.0 A resolution on a synchrotron radiation, and a full data set was collected at resolution of 2.0 A at 100K.c

Novel squalene-producing thraustochytrids found in mangrove water.

On extended screening of squalene-producing strains in Okinawa mangrove water, we identified 14 novel squalene-producing thraustochytrids from 172 unialgal clonal isolates. The novel thraustochytrids produced 13.9–7.54 mg squalene/g dry cell weight. Eight isolates were found to belong to potentially novel squalene-producing genera, forming a monophyletic cluster independent from any known thraustochytrids.

Trp residue at subsite − 5 plays a critical role in the substrate binding of two protistan GH26 β-mannanases from a termite hindgut

Symbiotic protists in the hindgut of termites provide a novel enzymatic resource for efficient lignocellulytic degradation of plant biomass. In this study, two β-mannanases, RsMan26A and RsMan26B, from a symbiotic protist community of the lower termite, Reticulitermes speratus, were successfully expressed in the methylotrophic yeast, Pichia pastoris. Biochemical characterization experiments demonstrated that both RsMan26A and RsMan26B are endo-acting enzymes and have a very similar substrate specificity, displaying a higher catalytic efficiency to galactomannan from locust bean gum (LBG) and glucomannan than to β-1,4-mannan and highly substituted galactomannan from guar gum. Homology modeling of RsMan26A and RsMan26B revealed that each enzyme displays a long open cleft harboring a unique hydrophobic platform (Trp79) that stacks against the sugar ring at subsite − 5. The Km values of W79A mutants of RsMan26A and RsMan26B to LBG increased by 4.8-fold and 3.6-fold, respectively, compared with those for the native enzymes, while the kcat remained unchanged or about 40% of that of the native enzyme, resulting in the decrease in the catalytic efficiency by 4.8-fold and 9.1-fold, respectively. The kinetic values for glucomannan also showed a similar result. These results demonstrate that the Trp residue present near the subsite − 5 has an important role in the recognition of the sugar ring in the substrate.