Levels of D-serine in the brain and peripheral organs of serine racemase (Srr) knock-out mice

d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor, plays an important role in mammalian brain neurotransmission, via the NMDA receptor. d-Serine is synthesized from l-serine by the pyridoxal-5′ phosphate-dependent enzyme serine racemase (SRR), and d-serine is metabolized by d-amino acid oxidase (DAAO). In this study, we measured levels of the neurotransmission related amino acids, d-serine, l-serine, glycine, glutamine and glutamate in the frontal cortex, hippocampus, striatum and cerebellum as well as in peripheral tissues of blood, heart, pancreas, spleen, liver, kidney, testis, epididymis, heart, lung, muscle and eyeball, in wild-type (WT) and Srr-knockout (Srr-KO) mice. Levels of d-serine in the frontal cortex, hippocampus, and striatum of Srr-KO mice were significantly lower than in WT mice, while levels in the cerebellum stayed the same. In contrast, levels of l-serine, glycine, glutamine and glutamate remained the same in all tested brain regions. In vivo microdialysis using free-moving mice showed that extracellular levels of d-serine in the hippocampus of Srr-KO mice were significantly lower than in WT mice while the other amino acid levels remained the same between mice. In peripheral organs, levels of d-serine in the kidney, testis, and muscle of Srr-KO mice were significantly lower than in WT mice. Tissue levels of the other tested amino acids in peripheral organs were not altered. These results suggest that SRR is the major enzyme responsible for d-serine production in the mouse forebrain, and that other pathways of d-serine production may exist in the brain and peripheral organs.     

A MOFRL family glycerate kinase from the thermophilic crenarchaeon, <Emphasis Type="Italic">Sulfolobus tokodaii</Emphasis>, with unique enzymatic properties

A glycerate kinase gene (ST2037) from the hyperthermophilic crenarchaeon Sulfolobus tokodaii was cloned and expressed in Escherichia coli. The purified homodimeric protein (45 kDa) specifically catalyzed the formation of 2-phosphoglycerate with d-glycerate as substrate. The thermostable enzyme displayed maximum activity (over 20 min) at 90°C and pH 4.5. The maximal activity was in the presence of Co²⁺. The MOFRL family glycerate kinase used AMP as phosphate donor with maximal activity towards GTP. These characteristics of the enzyme suggested its potential in the catalytic production of 2-phosphoglycerate.     

Purification,characterization, and gene cloning of a novel aminoacylase from Burkholderia sp. strain LP5_18B that efficiently catalyzes the synthesis of N-lauroyl-l-amino acids

ABSTRACT

An N-lauroyl-l-phenylalanine-producing bacterium, identified as Burkholderia sp. strain LP5_18B, was isolated from a soil sample. The enzyme was purified from the cell-free extract of the strain and shown to catalyze degradation and synthesis activities toward various N-acyl-amino acids. N-lauroyl-l-phenylalanine and N-lauroyl-l-arginine were obtained with especially high yields (51% and 89%, respectively) from lauric acid and l-phenylalanine or l-arginine by the purified enzyme in an aqueous system. The gene encoding the novel aminoacylase was cloned from Burkholderia sp. strain LP5_18B and expressed in Escherichia coli. The gene contains an open reading frame of 1,323 nucleotides. The deduced protein sequence encoded by the gene has approximately 80% amino acid identity to several hydratase of Burkholderia. The addition of zinc sulfate increased the aminoacylase activity of the recombinant E. coli strain.     

Preparation of Enzymes Required for Enzymatic Quantification of 5-Keto-D-gluconate and 2-Keto-D-gluconate

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

Characterization of Mesorhizobium loti L-Rhamnose Isomerase and Its Application to L-Talose Production

The L-rhamnose isomerase gene (rhi) of Mesorhizobium loti was cloned and expressed in Escherichia coli, and then characterized. The enzyme exhibited activity with respect to various aldoses, including D-allose and L-talose. Application of it in L-talose production from galactitol was achieved by a two-step reaction, indicating that it can be utilized in the large-scale production of L-talose.     

D-ribose-5-phosphate isomerase B from Escherichia coli is also a functional D-allose-6-phosphate isomerase, while the Mycobacterium tuberculosis enzyme is not

Interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate is an important step in the pentose phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars d-allose-6-phosphate (All6P) and d-allulose-6-phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme's primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-d-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme's structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate ribose-5-phosphate, shed additional light on the reaction mechanism of RpiBs generally.     

The ‘true’ l-xylulose reductase of filamentous fungi identified in Aspergillus niger

l-Xylulose reductase is part of the eukaryotic pathway for l-arabinose catabolism. A previously identified l-xylulose reductase in Hypocrea jecorina turned out to be not the ‘true’ one since it was not upregulated during growth on l-arabinose and the deletion strain showed no reduced l-xylulose reductase activity but instead lost the d-mannitol dehydrogenase activity [17]. In this communication we identified the ‘true’ l-xylulose reductase in Aspergillus niger. The gene, lxrA (JGI177736), is upregulated on l-arabinose and the deletion results in a strain lacking the NADPH-specific l-xylulose reductase activity and having reduced growth on l-arabinose. The purified enzyme had a K_m for l-xylulose of 25 mM and a ν_max of 650 U/mg.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Crystal structure of YihS in complex with D-mannose: structural annotation of Escherichia coli and Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerase

The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-d-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an α₆/α₆-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose-ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (d-mannose) at 1.6 Å resolution. This enzyme-substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, β-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, α₆/α₆-barrel.     

Dielectric properties of two diastereoisomers of the arabinose and their equimolar mixture

Dielectric relaxation measurements were performed on two enantiomers, d- and l-arabinose and their equimolar mixture, and compared to dielectric data obtained for d-ribose. d-Arabinose differs from d-ribose by having the opposite configuration at C2. This study reveals that both d- and l- of arabinose exhibit α-relaxation peaks with the same shape for the same α-relaxation time τ_α, and the same steepness index for the T_g-scale T-dependence of τ_α. However, the two isomers have slightly different glass transition temperatures T_g’s, and their secondary γ-relaxation times also differ slightly from the previously observed γ-relaxation in d-ribose at the same temperature. However, when samples of both investigated monosaccharides are annealed at higher temperatures, their glass transition temperatures become nearly identical. This is an effect of the mutarotation process, which leads to the formation of pairs of the enantiomers and accordingly they should have the same physical properties. The width of the α-relaxation of d- and l-arabinose is broader than that of d-ribose, as reflected by the smaller stretch exponent in the Kohlrausch-Williams-Watts function used to fit the data of the former (β_KWW = 0.46 ± 0.01) than the latter (β_KWW = 0.55 ± 0.01). The width of the α-relaxation of racemic mixture of the d- and l-arabinose is slightly broader than that of the pure isomers. While the dielectric loss data of d-ribose in the glassy state at ambient and elevated pressures show an inflexion indicating the presence of the JG β-relaxation, the data of d- and l-arabinose show no such feature for identification of the supposedly universal JG β-relaxation. Nevertheless, on comparing the loss spectra of d-arabinose with that of d-ribose, the presence of the JG β-relaxation in d-arabinose has been rationalized.     

A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292

4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O₂ or a cofactor for the conversion, but was stimulated by Mn²⁺. N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.     

Purification and properties of alanine racemase from crayfish Procambarus clarkii

Fresh water crayfish Procambarus clarkii is known to accumulate d-alanine remarkably in muscle after seawater acclimation, accompanied by an increase in alanine racemase activity. We have purified alanine racemase from crayfish muscle to homogeneity. The enzyme is a monomeric protein with a molecular mass of 58 kDa. It is highly specific to alanine and does not racemize l-serine, l-aspartate, l-glutamate, l-valine and l-arginine. The enzyme shows the highest activity at pH 9.0 in the conversion of l- to d-alanine and at pH 8.5 in the reverse conversion. Properties such as amino acid sequence, quaternary structure, pyridoxal 5′-phosphate (PLP)-dependency, pH-dependency and kinetic parameters seem to be distinct from those of the microbial alanine racemases. Various salts including NaCl at concentrations around seawater level were potently inhibitory for the activity in both of l- to -d and d- to -l direction.     

Bioconversion of ginsenosides Rb(1), Rb(2), Rc and Rd by novel β-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: cloning, expression, and enzyme characterization

A new β-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344 bp (447 amino acid residues) with a predicted molecular mass of 49,399 Da. A BLAST search using the bglSp sequence revealed significant homology to that of glycoside hydrolase superfamily 1. This enzyme was overexpressed in Escherichia coli BL21 (DE3) using a pET21-MBP (TEV) vector system. Overexpressed recombinant enzymes which could convert the ginsenosides Rb₁, Rb₂, Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc₁ and ginsenoside F₂, respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for β-glucosidase showed the apparent K_m and V_max values of 2.9 ± 0.3 mM and 515.4 ± 38.3 μmol min⁻¹ mg of protein⁻¹ against p-nitrophenyl-β-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb₁, Rb₂, Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc₁ and F₂ quickly at optimal conditions of pH 5.0 and 37 °C. A little ginsenoside F₂ production from ginsenosides Gyp XVII, C-O, and C-Mc₁ was observed for the lengthy enzyme reaction caused by the side ability of the enzyme.     

The structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-rhamnose and D-allose provide insights into broad substrate specificity

Pseudomonas stutzeril-rhamnose isomerase (P. stutzeri L-RhI) can efficiently catalyze the isomerization between various aldoses and ketoses, showing a broad substrate specificity compared to L-RhI from Escherichia coli (E. coli L-RhI). To understand the relationship between structure and substrate specificity, the crystal structures of P. stutzeri L-RhI alone and in complexes with l-rhamnose and d-allose which has different configurations of C4 and C5 from l-rhamnose, were determined at a resolution of 2.0 Å, 1.97 Å, and 1.97 Å, respectively. P. stutzeri L-RhI has a large domain with a (β/α)₈ barrel fold and an additional small domain composed of seven α-helices, forming a homo tetramer, as found in E. coli L-RhI and d-xylose isomerases (D-XIs) from various microorganisms. The β1-α1 loop (Gly60-Arg76) of P. stutzeri L-RhI is involved in the substrate binding of a neighbouring molecule, as found in D-XIs, while in E. coli L-RhI, the corresponding β1-α1 loop is extended (Asp52-Arg78) and covers the substrate-binding site of the same molecule. The complex structures of P. stutzeri L-RhI with l-rhamnose and d-allose show that both substrates are nicely fitted to the substrate -binding site. The part of the substrate-binding site interacting with the substrate at the 1, 2, and 3 positions is equivalent to E. coli L-RhI, and the other part interacting with the 4, 5, and 6 positions is similar to D-XI. In E. coli L-RhI, the β1-α1 loop creates an unique hydrophobic pocket at the the 4, 5, and 6 positions, leading to the strictly recognition of l-rhamnose as the most suitable substrate, while in P. stutzeri L-RhI, there is no corresponding hydrophobic pocket where Phe66 from a neighbouring molecule merely forms hydrophobic interactions with the substrate, leading to the loose substrate recognition at the 4, 5, and 6 positions.     

Gene Cloning and Characterization of α-Amino Acid Ester Acyl Transferase in Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458

The gene encoding α-amino acid ester acyl transferase (AET), the enzyme that catalyzes the peptide-forming reaction from amino acid methyl esters and amino acids, was cloned from Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458 and expressed in Escherichia coli. This is the first report on the aet gene. It encodes a polypeptide composed of 616 (ATCC14234) and 619 (AJ2458) amino acids residues. The V _max values of these recombinant enzymes during the catalysis of L-alanyl-L-glutamine formation from L-alanine methylester and L-glutamine were 1,010 U/mg (ATCC14234) and 1,154 U/mg (AJ2458). An amino acid sequence similarity search revealed 35% (ATCC14234) and 36% (AJ2458) identity with an α-amino acid ester hydrolase from Acetobacter pasteurianus, which contains an active-site serine in the consensus serine enzyme motif, GxSYxG. In the deduced amino acid sequences of AET from both bacteria, the GxSYxG motif was conserved, suggesting that AET is a serine enzyme.     

Cyclitols protect glutamine synthetase and malate dehydrogenase against heat induced deactivation and thermal denaturation

The accumulation of cyclitols in plants is a widespread response that provides protection against various environmental stresses. The capacity of myo-Inositol, pinitol, quercitol, and other compatible solutes (i.e., sorbitol, proline, and glycinebetaine) to protect proteins against thermally induced denaturation and deactivation was examined. Enzymatic activity measurements of l-glutamine synthetase from Escherichia coli and Hordeum vulgare showed that the presence of cyclitols during heat treatment resulted in a significantly higher percentage of residual activity. CD spectroscopy experiments were used to study thermal stabilities of protein secondary structures upon the addition of myo-Inositol, pinitol, and glucose. 0.4 M myo-Inositol was observed to raise the melting temperature (T_m) of GS from E. coli by 3.9 °C and MDH from pig heart by 3.4 °C, respectively. Pinitol showed an increase in T_m of MDH by 3.8 °C, whereas glucose was not effective. Our results show a great potential of stabilizing proteins by the addition of cyclitols.     

Analysis of the Hyperthermophilic Chitinase from Pyrococcus furiosus: Activity toward Crystalline Chitin

Chitinase [EC 3.2.1.14] is an enzyme that can hydrolyze the β-1,4 linkage between N-acetyl-D-glucosamine in chitin. In the genome database of the hyperthermophilic archaeon Pyrococcus furiosus, we found two adjacent genes (PF1233 and PF1234) homologous to those of the chitinase of Thermococcus kodakaraensis. In the cultured medium of P. furiosus, however, no chitinase activity was detected. On analysis of the structural gene of P. furiosus, it appears that one nucleotide insertion in PF1234 caused a frame shift and separated a gene. By deletion of one nucleotide in PF1234, the best match was achieved between chitinases of T. kodakaraenesis and P. furiosus. We succeeded in constructing an artificial recombinant chitinase exhibiting hydrolytic activity toward not only colloidal but also crystalline chitins at high temperature. Furthermore, by analyzing the characteristics of the domains, a recombinant enzyme comprising two domains exhibiting high activity toward crystalline chitin was prepared.     

Determination of plasma and serum l-lysine using l-lysine ε-oxidase from Marinomonas mediterranea NBRC 103028

This article describes a successful application of l-lysine ε-oxidase (EC 1.4.3.20) for l-lysine determination. l-Lysine ε-oxidase was isolated from culture supernatant of Marinomonas mediterranea NBRC 103028^T and was used for l-lysine determination. Comparison of the characteristics of l-lysine ε-oxidase with l-lysine α-oxidase, a commercial enzyme used for l-lysine determination, suggests that the use of l-lysine ε-oxidase would be more valuable for the determination of l-lysine because of its selectivity and sensitivity, especially in samples with low l-lysine concentration. The enzyme acted only on l-lysine and l-ornithine, to which the relative activity was only 3.4% of that on l-lysine. The value obtained by the colorimetric assay using l-lysine ε-oxidase and horseradish peroxidase was not affected by l-ornithine. The enzyme also shows a higher affinity for l-lysine (K_m = 0.0018 mM). l-Lysine determination using l-lysine ε-oxidase in human plasma and serum was examined. The measured values were close to values determined by instrumental analyses using the precolumn AccQ·Tag Ultra Derivatization Kit. These results suggest that l-lysine ε-oxidase can be used for diagnosis based on plasma l-lysine concentration. This is the first report on the application of l-lysine ε-oxidase.     

Expression, purification, and characterization of exo-β-d-glucosaminidase of Aspergillus sp. CJ22-326 from Escherichia coli

An exo-β-d-glucosaminidase gene was cloned from Aspergillus sp. CJ22-326 and expressed in Escherichia coli. The purified protein showed an exo-chitosanase activity in a viscosimetric assay and TLC analysis. This is the first report on cloning of a gene encoding an Aspergillus sp. exo-β-d-glucosaminidase.