Characterization of hydantoin-5-propionic acid amidohydrolase involved in ergothioneine utilization in Burkholderia sp. HME13.

In our previous study, ertABC genes encoding ergothionase, thiourocanate hydratase, and 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase were identified, all of which may be involved in ergothioneine utilization of Burkholderia sp. HME13. In this study, we identify the ertD gene encoding metal-dependent hydantoin-5-propionic acid amidohydrolase in this strain. Mn2+-containing ErtD showed maximum activity at 45 °C and pH 8.5 and was stable at temperatures up to 45 °C. The Km and Vmax values of Mn2+-containing ErtD for hydantoin-5-propionic acid were 2.8 m m and 16 U/mg, respectively. Real-time polymerase chain reaction (PCR) revealed that ertD expression levels in Burkholderia sp. HME13 cells cultivated in ergothioneine medium were 3.3-fold higher than those in cells cultivated in Luria-Bertani (LB) medium. ErtD activity in the crude extract from Burkholderia sp. HME13 cells cultured in ergothioneine medium was 0.018 U/mg, whereas that in LB medium was not detected. Accordingly, we suggest that ErtD is involved in ergothioneine utilization in this strain.

Purification and characterization of 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase involved in ergothioneine utilization in Burkholderia sp. HME13.

Recombinant 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase (ErtC) derived from Burkholderia sp. HME13 was purified to homogeneity. Here, ErtC's kinetic parameters, optimum reaction temperature and pH, and stability at varying temperatures and pH and the effects of various additives on ErtC activity were determined. Real-time polymerase chain reaction and enzyme assays suggested that ergothioneine induced the expression of ertC.

Identification of the gene encoding 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase in Burkholderia sp. HME13.

Here, we report the identification of the gene encoding a novel enzyme, 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase, in Burkholderia sp. HME13. The enzyme converts 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid and H2O to 3-(2,5-dioxoimidazolidin-4-yl) propionic acid and H2S. Amino acid sequence analysis of the enzyme indicates that it belongs to the DUF917 protein family, which consists of proteins of unknown function.

Characterization of an Alteromonas long-type ulvan lyase involved in the degradation of ulvan extracted from Ulva ohnoi.

Ulvan is a sulfated polysaccharide found in the cell wall of the green algae Ulva. We first isolated several ulvan-utilizing Alteromonas sp. from the feces of small marine animals. The strain with the highest ulvan-degrading activity, KUL17, was analyzed further. We identified a 55-kDa ulvan-degrading protein secreted by this strain and cloned the gene encoding for it. The deduced amino acid sequence indicated that the enzyme belongs to polysaccharide lyase family 24 and thus the protein was named ulvan lyase. The predicted molecular mass of this enzyme is 110 kDa, which is different from that of the identified protein. By deletion analysis, the catalytic domain was proven to be located on the N-terminal half of the protein. KUL17 contains two ulvan lyases, one long and one short, but the secreted and cleaved long ulvan lyase was demonstrated to be the major enzyme for ulvan degradation.

Characterization of ergothionase from Burkholderia sp. HME13 and its application to enzymatic quantification of ergothioneine.

We identified ergothionase, which catalyzes conversion of ergothioneine to thiolurocanic acid and trimethylamine, in a newly isolated ergothioneine-utilizing strain, Burkholderia sp. HME13. The enzyme was purified and its N-terminal amino acid sequence was determined. Based on the amino acid sequence, the gene encoding the enzyme was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity and characterized. The enzyme consisted of four identical 55-kDa subunits. The enzyme showed maximum activity at pH 8.0 and 65 °C and was stable between pH 7.0 and pH 10.0 and up to 60 °C. The enzyme acted on ergothioneine (K m: 19 µM, V max: 270 µmol/min/mg), but not D-histidine, L-histidine, D-tyrosine, L-tyrosine, D-phenylalanine, or L-phenylalanine. The enzyme was activated by BaCl2 and strongly inhibited by CuSO4, ZnSO4, and HgCl2. The amino acid sequence of ergothionase showed 23 % similarity to histidine ammonia-lyase (HAL) from Pseudomonas putida and 17 % similarity to phenylalanine ammonia-lyase (PAL) from parsley. However, the tripeptide sequence, Ala-Ser-Gly, which is important for catalysis in both HAL and PAL, was not conserved in ergothionase. The application of ergothionase for the quantification of ergothioneine contained in practical food and blood samples was investigated by performing a recovery test. Satisfactory recovery data (98.7-104 %) were obtained when ergothioneine was added to extract of tamogitake and hemolysis blood.

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.

Enzymatic determination of carnosine in meat and fish using ß-Ala-Xaa dipeptidase and histidine ammonia-lyase derived from Pseudomonas putida NBRC100650.

Carnosine is a naturally occurring dipeptide and a functional component in foods, while also showing health-promoting effects. Generally, food-derived carnosine is quantified via high-performance liquid chromatography (HPLC). We have developed a method for quantifying carnosine in foods using microbial enzymes, ß-Ala-Xaa dipeptidase (BapA) and histidine ammonia-lyase (HAL). The carnosine concentrations in extracts of chicken, pork, beef, bonito, and tuna were determined via both HPLC and enzymatic determination. The carnosine contents measured via enzymatic determination were in agreement with those determined via conventional HPLC analysis. Relative standard-deviation values of the conventional HPLC method and the enzymatic determination of carnosine in foods were 0.728-5.76% and 0.504-4.58%, respectively. The recovery of carnosine in food extracts via enzymatic determination was 97-103%. Therefore, the developed enzymatic determination technique using BapA and HAL can be used for the determination of carnosine in meats and fishes with comparable accuracy to that of conventional HPLC analysis.

Discovery and characterization of D-phenylserine deaminase from Arthrobacter sp. TKS1.

We discovered a D-phenylserine deaminase that catalyzed the pyridoxal 5'-phosphate (PLP)-dependent deamination reaction from D-threo-phenylserine to phenylpyruvate in newly isolated Arthrobacter sp. TKS1. The enzyme was partially purified, and its N-terminal amino acid sequence was analyzed. Based on the sequence information, the gene encoding the enzyme was identified and expressed in Escherichia coli. The expressed protein was purified to homogeneity and characterized. The enzyme consisted of two identical 46-kDa subunits and showed maximum activity at pH 8.5 and 55°C. The enzyme was stable in the range of pH 7.5 to pH 8.5 and up to 50°C. The enzyme acted on the D-forms of ß-hydroxy-α-amino acids, such as D-threo-phenylserine (K(m), 19 mM), D-serine (K(m), 5.8 mM), and D-threonine (K(m), 102 mM). As L-threonine, D-allo-threonine, L-allo-threonine, and DL-erythro-phenylserine were inert, the enzyme could distinguish D-threo-form from among the four stereoisomers of phenylserine or threonine. The enzyme was activated by ZnSO(4), CuSO(4), BaCl(2), and CoCl(2) and strongly inhibited by phenylhydrazine, sodium borohydride, hydroxylamine, and DL-penicillamine. The enzyme exhibited absorption maxima at 280 and around 415 nm. The enzyme has an N-terminal domain similar to that of alanine racemase, which belongs to the fold type III group of pyridoxal enzymes.

Gene cloning and characterization of thiourocanate hydratase from Burkholderia sp. HME13.

A novel enzyme, thiourocanate hydratase, which catalyses the conversion of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid, was isolated from the ergothioneine-utilizing strain, Burkholderia sp. HME13. When the HME13 cells were cultured in medium containing ergothioneine as the sole nitrogen source, thiourocanate-metabolizing activity was detected in the crude extract from the cells. However, activity was not detected in the crude extract from HME13 cells that were cultured in Luria-Bertani medium. The gene encoding thiourocanate hydratase was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme showed maximum activity at pH 7.5 and 55°C and was stable between pH 5.0 and 10.5, and at temperatures up to 45°C. The Km and Vmax values of thiourocanate hydratase towards thiourocanic acid were 30 µM and 7.1 µmol/min/mg, respectively. The enzyme was strongly inhibited by CuCl2 and HgCl2. The amino acid sequence of the enzyme showed 46% identity to urocanase from Pseudomonas putida, but thiourocanate hydratase had no urocanase activity.

Prediction of missing enzyme genes in a bacterial metabolic network. Reconstruction of the lysine-degradation pathway of Pseudomonas aeruginosa.

The metabolic network is an important biological network which consists of enzymes and chemical compounds. However, a large number of metabolic pathways remains unknown, and most organism-specific metabolic pathways contain many missing enzymes. We present a novel method to identify the genes coding for missing enzymes using available genomic and chemical information from bacterial genomes. The proposed method consists of two steps: (a) estimation of the functional association between the genes with respect to chromosomal proximity and evolutionary association, using supervised network inference; and (b) selection of gene candidates for missing enzymes based on the original candidate score and the chemical reaction information encoded in the EC number. We applied the proposed methods to infer the metabolic network for the bacteria Pseudomonas aeruginosa from two genomic datasets: gene position and phylogenetic profiles. Next, we predicted several missing enzyme genes to reconstruct the lysine-degradation pathway in P. aeruginosa using EC number information. As a result, we identified PA0266 as a putative 5-aminovalerate aminotransferase (EC 2.6.1.48) and PA0265 as a putative glutarate semialdehyde dehydrogenase (EC 1.2.1.20). To verify our prediction, we conducted biochemical assays and examined the activity of the products of the predicted genes, PA0265 and PA0266, in a coupled reaction. We observed that the predicted gene products catalyzed the expected reactions; no activity was seen when both gene products were omitted from the reaction.

N-methyl-L-amino acid dehydrogenase from Pseudomonas putida. A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily.

We found N-methyl-L-amino acid dehydrogenase activity in various bacterial strains, such as Pseudomonas putida and Bacillus alvei, and cloned the gene from P. putida ATCC12633 into Escherichia coli. The enzyme purified to homogeneity from recombinant E. coli catalyzed the NADPH-dependent formation of N-alkyl-L-amino acids from the corresponding alpha-oxo acids (e.g. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (e.g. methylamine, ethylamine, and propylamine). Ammonia was inert as a substrate, and the enzyme was clearly distinct from conventional NAD(P)-dependent amino acid dehydrogenases, such as alanine dehydrogenase (EC 1.4.1.1). NADPH was more than 300 times more efficient than NADH as a hydrogen donor in the enzymatic reductive amination. Primary structure analysis revealed that the enzyme belongs to a new NAD(P)-dependent oxidoreductase superfamily, the members of which show no sequence homology to conventional NAD(P)-dependent amino acid dehydrogenases and opine dehydrogenases.

A new family of NAD(P)H-dependent oxidoreductases distinct from conventional Rossmann-fold proteins.

A new family of NAD(P)H-dependent oxidoreductases is now recognized as a protein family distinct from conventional Rossmann-fold proteins. Numerous putative proteins belonging to the family have been annotated as malate dehydrogenase (MDH) or lactate dehydrogenase (LDH) according to the previous classification as type-2 malate/L-lactate dehydrogenases. However, recent biochemical and genetic studies have revealed that the protein family consists of a wide variety of enzymes with unique catalytic activities other than MDH or LDH activity. Based on their sequence homologies and plausible functions, the family proteins can be grouped into eight clades. This classification would be useful for reliable functional annotation of the new family of NAD(P)H-dependent oxidoreductases.

Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15.

The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3) were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD(+)-dependent oxidation of the ß-hydroxyl group of l-ß-phenylserine. l-Phenylserine and l-threo-(2-thienyl)serine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO(2).

Enzymatic synthesis of L-pipecolic acid by Delta1-piperideine-2-carboxylate reductase from Pseudomonas putida.

L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine alpha-oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Delta1-piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of L-pipecolic acid in 99.7% e.e.

The putative malate/lactate dehydrogenase from Pseudomonas putida is an NADPH-dependent delta1-piperideine-2-carboxylate/delta1-pyrroline-2-carboxylate reductase involved in the catabolism of D-lysine and D-proline.

A Pseudomonas putida ATCC12633 gene, dpkA, encoding a putative protein annotated as malate/L-lactate dehydrogenase in various sequence data bases was disrupted by homologous recombination. The resultant dpkA(-) mutant was deprived of the ability to use D-lysine and also D-proline as a sole carbon source. The dpkA gene was cloned and overexpressed in Escherichia coli, and the gene product was characterized. The enzyme showed neither malate dehydrogenase nor lactate dehydrogenase activity but catalyzed the NADPH-dependent reduction of such cyclic imines as Delta(1)-piperideine-2-carboxylate and Delta(1)-pyrroline-2-carboxylate to form L-pipecolate and L-proline, respectively. NADH also served as a hydrogen donor for both substrates, although the reaction rates were less than 1% of those with NADPH. The reverse reactions were also catalyzed by the enzyme but at much lower rates. Thus, the enzyme has dual metabolic functions, and we named the enzyme Delta(1)-piperideine-2-carboxylate/Delta(1)-pyrroline-2-carboxylate reductase, the first member of a novel subclass in a large family of NAD(P)-dependent oxidoreductases.

Crystal structures of Delta1-piperideine-2-carboxylate/Delta1-pyrroline-2-carboxylate reductase belonging to a new family of NAD(P)H-dependent oxidoreductases: conformational change, substrate recognition, and stereochemistry of the reaction.

Delta(1)-Piperideine-2-carboxylate/Delta(1)-pyrroline-2-carboxylate reductase from Pseudomonas syringae pv. tomato belongs to a novel sub-class in a large family of NAD(P)H-dependent oxidoreductases distinct from the conventional MDH/LDH superfamily characterized by the Rossmann fold. We have determined the structures of the following three forms of the enzyme: the unliganded form, the complex with NADPH, and the complex with NADPH and pyrrole-2-carboxylate at 1.55-, 1.8-, and 1.7-A resolutions, respectively. The enzyme exists as a dimer, and the subunit consists of three domains; domain I, domain II (NADPH binding domain), and domain III. The core of the NADPH binding domain consists of a seven-stranded predominantly antiparallel beta-sheet fold (which we named SESAS) that is characteristic of the new oxidoreductase family. The enzyme preference for NADPH over NADH is explained by the cofactor binding site architecture. A comparison of the overall structures revealed that the mobile domains I and III change their conformations to produce the catalytic form. This conformational change plays important roles in substrate recognition and the catalytic process. The active site structure of the catalytic form made it possible to identify the catalytic Asp:Ser:His triad and investigate the catalytic mechanism from a stereochemical point of view.