Structure of pyridoxal 5'-phosphate-bound D-threonine aldolase from Chlamydomonas reinhardtii.

D-Threonine aldolase (DTA) is a pyridoxal-5'-phosphate-dependent enzyme which catalyzes the reversible aldol reaction of glycine with a corresponding aldehyde to yield the D-form ß-hydroxy-α-amino acid. This study produced and investigated the crystal structure of DTA from Chlamydomonas reinhardtii (CrDTA) at 1.85 Šresolution. To our knowledge, this is the first report on the crystal structure of eukaryotic DTA. Compared with the structure of bacterial DTA, CrDTA has a similar arrangement of active-site residues. On the other hand, we speculated that some non-conserved residues alter the affinity for substrates and inhibitors. The structure of CrDTA could provide insights into the structural framework for structure-guided protein engineering studies to modify reaction selectivity.

Rapid removal of detergent in glycolipids using ionic liquids.

Detergent removal in glycolipid after sample preparation, such as enzymatic reaction or isolation of detergent-resistant membrane microdomain, is indispensable for further structural characterization. We previously established the rapid and effective method of detergent removal in glycolipid samples from glass test tube using 1,2-dichloroethane (DCE) washing. However, the use of DCE has several drawbacks, such as environmental risks, harmful effects (potentially carcinogenic), and high vaporability and flammability. To solve the issue, we used ionic liquids to remove detergents from glycolipid samples, and found 1-butyl-3-methylimidazolium iodide was a suitable alternative for DCE.

Involvement of C-terminal amino acids of a hyperthermophilic serine racemase in its thermostability.

Pyrobaculum islandicum is a hyperthermophilic archaeon that grows optimally at 95-100 °C. In the previous study, we extensively purified a serine racemase from this organism and cloned the gene for overexpression in Escherichia coli (Ohnishi et al. 2008). This enzyme also exhibits highly thermostable L-serine/L-threonine dehydratase activity. In the present study, we aimed to elucidate the molecular mechanisms underlying the high thermostability of this enzyme. A recombinant variant of this enzyme, PiSRvt, constructed by truncating the C-terminal 72 amino acids, was compared with the native enzyme, PiSR. The dehydratase activity of PiSR and PiSRvt was found to owe to a homotrimer and a monomer, respectively, that demonstrated high and moderate thermostability, respectively. These observations reveal that the C-terminal region contributes to monomer trimerization that provides the extreme thermostability.

Crystallization and X-ray analysis of D-threonine aldolase from Chlamydomonas reinhardtii.

D-Threonine aldolase from the green alga Chlamydomonas reinhardtii (CrDTA) catalyzes the interconversion of several ß-hydroxy-D-amino acids (e.g. D-threonine) and glycine plus the corresponding aldehydes. Recombinant CrDTA was overexpressed in Escherichia coli and purified to homogeneity; it was subsequently crystallized using the hanging-drop vapour-diffusion method at 295 K. Data were collected and processed at 1.85 Šresolution. Analysis of the diffraction pattern showed that the crystal belonged to space group P1, with unit-cell parameters a = 64.79, b = 74.10, c = 89.94 Å, α = 77.07, ß = 69.34, γ = 71.93°. The asymmetric unit contained four molecules of CrDTA. The Matthews coefficient was calculated to be 2.12 Å3 Da-1 and the solvent content was 41.9%.

Cloning and characterization of d-threonine aldolase from the green alga Chlamydomonas reinhardtii.

d-Threonine aldolase (DTA) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent interconversion of d-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various ß-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70 °C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50 °C for 1 h. The enzyme catalyzed the synthesis of d- and d-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of d-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors.

Purification of Pyridylaminated Oligosaccharides Using 1,2-Dichloroethane Extraction.

Fluorescence derivatization of the oligosaccharides released from glycoconjugates is widely used for precise structural characterization. To ensure labeling of the oligosaccharides, a large excess of fluorescence reagents is usually added to the reaction tube. Therefore, any excess reagents and by-products of the labeling reaction should be removed by several column chromatographies, including using a cellulose cartridge or spin columns. However, these purification steps are often time-consuming, expensive, and laborious. In this study, we found that 1,2-dichloroethane extraction could effectively and easily purify pyridylaminated oligosaccharides with a high recovery rate.

Purification of Helicobacter pylori NCTC 11637 cytochrome bc1 and respiration with D-proline as a substrate.

Helicobacter pylori is a microaerophilic bacterium associated with gastric inflammation and peptic ulcers. Knowledge of how pathogenic organisms produce energy is important from a therapeutic point of view. We found d-amino acid dehydrogenase-mediated electron transport from d-proline or d-alanine to oxygen via the respiratory chain in H. pylori. Coupling of the electron transport to ATP synthesis was confirmed by using uncoupler reagents. We reconstituted the electron transport chain to demonstrate the electron flow from the d-amino acids to oxygen using the recombinant cytochrome bc(1) complex, cytochrome c-553, and the terminal oxidase cytochrome cbb(3) complex. Upon addition of the recombinant d-amino acid dehydrogenase and d-proline or d-alanine to the reconstituted electron transport system, reduction of cytochrome cbb(3) and oxygen consumption was revealed spectrophotometrically and polarographically, respectively. Among the constituents of H. pylori's electron transport chain, only the cytochrome bc(1) complex had been remained unpurified. Therefore, we cloned and sequenced the H. pylori NCTC 11637 cytochrome bc(1) gene clusters encoding Rieske Fe-S protein, cytochrome b, and cytochrome c(1), with calculated molecular masses of 18 kDa, 47 kDa, and 32 kDa, respectively, and purified the recombinant monomeric protein complex with a molecular mass of 110 kDa by gel filtration. The absorption spectrum of the recombinant cytochrome bc(1) complex showed an alpha peak at 561 nm with a shoulder at 552 nm.

D-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637.

Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. D-Amino acid dehydrogenase is a flavoenzyme that digests free neutral D-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 D-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial D-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to D-proline. The enzyme mediated electron transport from D-proline to coenzyme Q(1), thus distinguishing it from D-amino acid oxidase. The apparent K(m) and V(max) values were 40.2 mM and 25.0 micromol min(-1) mg(-1), respectively, for dehydrogenation of D-proline, and were 8.2 microM and 12.3 micromol min(-1) mg(-1), respectively, for reduction of Q(1). The respective pH and temperature optima were 8.0 and 37 degrees C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian D-amino acid oxidase than other bacterial D-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from D-proline to a c-type cytochrome was suggested spectrophotometrically.

Purification and characterization of serine racemase from a hyperthermophilic archaeon, Pyrobaculum islandicum.

Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100 degrees C. A pyridoxal 5'-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high L-serine/L-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95 degrees C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either L-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and Vmax values of the racemase activity of Srr for L-serine were 185 mM and 20.1 micromol/min/mg, respectively, while the corresponding values of the dehydratase activity of L-serine were 2.2 mM and 80.4 micromol/min/mg, respectively.

Alanine racemase from Helicobacter pylori NCTC 11637: purification, characterization and gene cloning.

The Helicobacter pylori NCTC 11637 alanine racemase gene, alr1, was cloned based on a putative alanine racemase gene, alr, of H. pylori 26695. The protein, Alr1, was purified to homogeneity from Escherichia coli MB2795 cells harboring the alr1 gene. The protein exclusively catalyzes the conversion of l-alanine to the d-isomer with K(m) and V(max) values of 100 mM and 909 mumol min(-1) mg(-1), respectively. The values are 16-fold higher than those for the reaction in the reverse direction. The molecular weight of Alr1 is 42,000 by SDS-PAGE, and 68,000 by gel-filtration analysis. The optimal pH and temperature are pH 8.3 and 37 degrees C, respectively, in good accordance with the characteristics shown by the alanine racemase purified from H. pylori NCTC 11637 cells. Pyridoxal 5'-phosphate was suggested to be the cofactor. The physiological function of Alr1 is discussed regarding energy production in the microbial cells.

Identification of enzyme responsible for erythritol utilization and reaction product in yeast Lipomyces starkeyi.

We have identified the enzyme responsible for erythritol utilization and its reaction product in the yeast Lipomyces starkeyi CBS 1807. The enzyme, a polyol dehydrogenase requiring NAD+ as a coenzyme, was induced by erythritol in this yeast. We confirmed that the enzyme product was L-erythrulose by MS, NMR, and polarimeter analyses, meaning that we clarified the first step of erythritol utilization in yeasts for the first time. In the case of the oxidative reaction, D-threitol, (2R,3R)-2,3-butanediol, and erythritol were much better substrates than 21 other polyols tested. These three substrates are tetroses and have an R configuration at C-3, and whose third carbon results in easiest oxidation in this enzyme. The research of the substrate specificity in the reductive reaction demonstrated that L-erythrulose and dihydroxyacetone were better substrates, that D-acetoin was inactive and L-erythrose (aldose) was slightly active.

Total DNA preparation excluding extracellular acidic polysaccharide from Lipomyces yeasts and its application to taxonomic studies.

A preparation method of total DNA from Lipomyces yeasts was improved in order to exclude extracellular acidic polysaccharide thoroughly. The method combined an ultracentrifuge and polyethylene glycol precipitation with the usual method. The total DNAs obtained were analyzed for G + C content and by DNA-DNA hybridization. The results all agreed almost completely with literature data. All the DNA samples prepared using this method were pure enough for these taxonomic analyses and could also be used as templates of PCR for amplification of small subunit ribosomal DNA and the internal transcribed spacer region.

Development of a new chemically defined sporulation medium for the yeasts in the genus Lipomyces.

A chemically defined sporulation medium (AF medium) for the yeasts belonging to the genus Lipomyces was developed. The chemical composition was derived from chemical analyses of soybean extract. Some chemical modification of the AF medium indicated that the nitrogen sources (aspartic and glutamic acids) and zinc ion were essential for sporulation. The significance of medium pH was discussed.