Daisuke Matsui

A periplasmic, pyridoxal-5′-phosphate-dependent amino acid racemase in Pseudomonas taetrolens

The pyridoxal-5′-phosphate (PLP)-dependent amino acid racemases occur in almost every bacterium but may differ considerably with respect to substrate specificity. We here isolated the cloned broad substrate specificity racemase ArgR of Pseudomonas taetrolens from Escherichia coli by classical procedures. The racemase was biochemically characterized and amongst other aspects it was confirmed that it is mostly active with lysine, arginine and ornithine, but merely weakly active with alanine, whereas the alanine racemase of the same organism studied in comparison acts on alanine only. Unexpectedly, sequencing the amino-terminal end of ArgR revealed processing of the protein, with a signal peptide cleaved off. Subsequent localization studies demonstrated that in both P. taetrolens and E. coli ArgR activity was almost exclusively present in the periplasm, a feature so far unknown for any amino acid racemase. An ArgR-derivative carrying a carboxy-terminal His-tag was made and this was demonstrated to localize even in an E. coli mutant devoid of the twin-arginine translocation (Tat) pathway in the periplasm. These data indicate that ArgR is synthesized as a prepeptide and translocated in a Tat-independent manner. We therefore propose that ArgR translocation depends on the Sec system and a post-translocational insertion of PLP occurs. As further experiments showed, ArgR is necessary for the catabolism of d-arginine and d-lysine by P. taetrolens.

Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate

BackgroundCorynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032.ResultsHere, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer.ConclusionsCg1067 encodes quinone-dependent D-lactate dehydrogenase Dld of Corynebacterium glutamicum. Dld is essential for growth with D-lactate as sole carbon source. The genomic region of dld likely has been acquired by horizontal gene transfer.

Characterization of a pyridoxal-5′-phosphate-dependent l-lysine decarboxylase/oxidase from Burkholderia sp. AIU 395

A novel enzyme, which catalyzed decarboxylation of l-lysine into cadaverine with release of carbon dioxide and oxidative deamination of l-lysine into l-2-aminoadipic 5-semialdehyde with release of ammonia and hydrogen peroxide, was found from a newly isolated Burkholderia sp. AIU 395. The enzyme was specific to l-lysine and did not exhibit enzyme activities for other l-amino acids, l-lysine derivatives, d-amino acids, and amines. The apparent Km values for l-lysine in the oxidation and decarboxylation reactions were estimated to be 0.44xa0mM and 0.84xa0mM, respectively. The molecular mass was estimated to be 150xa0kDa, which was composed of two identical subunits with molecular mass of 76.5xa0kDa. The enzyme contained one mol of pyridoxal 5-phosphate per subunit as a prosthetic group. The enzyme exhibiting decarboxylase and oxidase activities for l-lysine was first reported here, while the deduced amino acid sequence was homologous to that of putative lysine decarboxylases from the genus Burkholderia.

Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities

In this study, it was shown for the first time that l‐amino acid oxidase of Pseudomonas sp. AIU813, renamed as l‐amino acid oxidase/monooxygenase (l‐AAO/MOG), exhibits l‐lysine 2‐monooxygenase as well as oxidase activity. l‐Lysine oxidase activity of l‐AAO/MOG was increased in a p‐chloromercuribenzoate (p‐CMB) concentration‐dependent manner to a final level that was five fold higher than that of the non‐treated enzyme. In order to explain the effects of modification by the sulfhydryl reagent, saturation mutagenesis studies were carried out on five cysteine residues, and we succeeded in identifying l‐AAO/MOG C254I mutant enzyme, which showed five‐times higher specific activity of oxidase activity than that of wild type. The monooxygenase activity shown by the C254I variant was decreased significantly. Moreover, we also determined a high‐resolution three‐dimensional structure of l‐AAO/MOG to provide a structural basis for its biochemical characteristics. The key residue for the activity conversion of l‐AAO/MOG, Cys‐254, is located near the aromatic cage (Trp‐418, Phe‐473, and Trp‐516). Although the location of Cys‐254 indicates that it is not directly involved in the substrate binding, the chemical modification by p‐CMB or C254I mutation would have a significant impact on the substrate binding via the side chain of Trp‐516. It is suggested that a slight difference of the binding position of a substrate can dictate the activity of this type of enzyme as oxidase or monooxygenase.

Detection of D-Ornithine Extracellularly Produced by Corynebacterium glutamicum ATCC 13032: argF

We found that Corynebacterium glutamicum ATCC 13032::argF extracellularly produced a large amount of D-ornithine when cultivated in a CGXII medium containing 1 mM L-arginine. This is the first report that C. glutamicum ATCC 13032 or its mutant produces a D-amino acid extracellularly. C. glutamicum ATCC 13032::argF produced 13 mM D-ornithine in 45 h of cultivation.

Characterization of two amine oxidases from Aspergillus carbonarius AIU 205.

We have reported that Aspergillus carbonarius AIU 205, which was isolated by our group, produced three enzymes exhibiting oxidase activity for 4-aminobutanamide (4-ABAD) (J. Biosci. Bioeng., 117, 263-268, 2014). Among three enzymes, characteristics of enzyme I have been revealed, but those of the other two enzymes have not. In this study, we purified enzymes II and III, and compared their characteristics with those of enzyme I. Enzymes II and III also oxidized aliphatic monoamines, aromatic amines, and aliphatic aminoalcohols. In addition, the oxidase activity of both enzymes was strongly inhibited by carbonyl reagents and specific inhibitors for copper-containing amine oxidases. Thus, enzymes II and III were also classified into the copper-containing amine oxidase group (EC 1.4.3.6) along with enzyme I. However, these three enzymes differed from each other in their enzymatic, kinetic, and physicochemical properties. The N-terminal amino acid sequences also differed from each other; that of enzyme I was modified, that of enzyme II was similar to those of peroxisomal copper-containing amine oxidases, and that of enzyme III was similar to those of copper-containing amine oxidases from the genus Aspergillus. Therefore, we concluded that A.xa0carbonarius AIU 205 produced three different types of amine oxidase in the mycelia.

Detection and Function of the Intramolecular Disulfide Bond in Arginine Racemase: An Enzyme with Broad Substrate Specificity

We found that a single intramolecular disulfide bond between the cysteines C47 and C73 exists in the primary structure of arginine racemase (ArgR) from Pseudomonas taetrolens NBRC 3460, and this is the first example of a pyridoxal phosphate (PLP)‐dependent amino acid racemase that contains a disulfide bond. The amino acid racemase activity was still detected, when the disulfide bond of ArgR was disrupted by site‐directed mutagenesis or reduced with dithiothreitol (DTT). The thermal and pH profiles and the quaternary structure of ArgR did not change when the disulfide bond of ArgR was disrupted by site‐directed mutagenesis. The substrate specificity and the overall structure did not change when the disulfide bond of ArgR was reduced with DTT after the protein was matured. However, these properties changed when the disulfide bond of ArgR was disrupted by site‐directed mutagenesis before protein maturation. The total activity of ArgR decreased when the disulfide bond of ArgR was disrupted by site‐directed mutagenesis before the protein was matured or when ArgR was expressed in the cytoplasm. Based on these results, we can conclude that the disulfide bond of ArgR is essential for ArgR to fold and mature as an amino acid racemase with broad substrate specificity.

Association between α-Klotho and Deep White Matter Lesions in the Brain: A Pilot Case Control Study Using Brain MRI

BACKGROUNDnThe anti-aging protein, α-Klotho, may be involved in cognitive decline and has potential as a surrogate marker that reflects dementia. However, the role of α-Klotho in the brain has not been sufficiently investigated.nnnOBJECTIVEnHere, we investigated the association between α-Klotho and cognitive decline that is associated with cerebral deep white matter lesions (DWMLs).nnnMETHODSnTwo hundred-eighty participants (187 males and 93 females, mean age: 70.8 years old) were evaluated for DWMLs, and the Fazekas scale (Grade) was assessed following brain magnetic resonance imaging. A questionnaire concerning lifestyle and neuropsychological tests was administered, and their associations with the blood α-Klotho level were retrospectively investigated.nnnRESULTSnThe α-Klotho level was 685.1u200apg/mL in Grade 0 (68 subjects), 634.1 in G1 (134), 596.0 in G2 (62), and 571.6 in G3 (16), showing that the level significantly decreased with advanced grades. Significant correlations were noted between the α-Klotho level and higher brain function tests including the Mini-Mental State Examination and word fluency tests (pu200a<u200a0.05). When a 90th percentile value of the level in the G0 group (400u200apg/mL) or lower was defined as a low α-Klotho level, the odds ratio of the high-grade G3 group was 2.9 (95% confidence interval: 1.4-7.8) (after correction for age, sex, hypertension, and chronic kidney disease), which was significant.nnnCONCLUSIONnA reduced blood α-Klotho level was correlated with grading of cerebral DWMLs and was accompanied by cognitive decline as an independent risk factor. The α-Klotho level may serve as a useful clinical index of vascular cognitive impairment.

Enhancement of stability of l-tryptophan dehydrogenase from Nostoc punctiforme ATCC29133 and its application to l-tryptophan assay

Microbial NAD(+)-dependent L-tryptophan dehydrogenase (TrpDH, EC1.4.1.19), which catalyzes the reversible oxidative deamination and the reductive amination between L-tryptophan and indole-3-pyruvic acid, was found in the scytonemin biosynthetic pathway of Nostoc punctiforme ATCC29133. The TrpDH exhibited high specificity toward L-tryptophan, but its instability was a drawback for L-tryptophan determination. The mutant enzyme TrpDH L59F/D168G/A234D/I296N with thermal stability was obtained by screening of Escherichia coli transformants harboring various mutant genes, which were generated by error-prone PCR using complementation in an L-tryptophan auxotroph of E. coli. The specific activity and stability of this mutant enzyme were higher than those of the wild type enzyme. We also revealed here that in these four mutation points, the two amino acid residues Asp168 and Ile296 contributed to increase the enzyme stability, and the Leu59, Ala234 residues to increase its specific activity. Growth of the strain harboring the gene of above 4 point mutated enzyme was accelerated by the enhanced performance. In the present study, we demonstrated that TrpDH L59F/D168G/A234D/I296N was available for determination of L-tryptophan in human plasma.

New enzymatic methods for selective assay of L-lysine using an L-lysine specific decarboxylase/oxidase from Burkholderia sp. AIU 395.

We developed new enzymatic methods for the selective assay of L-lysine by utilizing an oxidase reaction and a decarboxylation reaction by the L-lysine-specific decarboxylase/oxidase (L-Lys-DC/OD) from Burkholderia sp. AIU 395. The method utilizing the oxidase reaction of this enzyme was useful for determination of high concentrations of L-lysine. The method utilizing the decarboxylase reaction, which proceeded via the combination of the L-Lys-DC/OD and putrescine oxidase (PUO) from Micrococcus rubens, was effective for determination of low concentrations of L-lysine. Both methods showed good linearity, and neither was affected by other amino acids or amines. In addition, the within-assay and between-assay precisions of both methods were within the allowable range. The coupling of L-Lys-DC/OD with PUO was also useful for the differential assay of L-lysine and cadaverine. These newly developed methods were applied to the assay of L-lysine in biological samples and found to be effective.