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NAD-dependent L-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of L-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as L-aspartate (1,735%) and L-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.

A novel alanine:glyoxylate aminotransferase was found in a hyperthermophilic archaeon, Thermococcus litoralis. The amino acid sequence of the enzyme did not show a similarity to any alanine:glyoxylate aminotransferases reported so far. Homologues of the enzyme appear to be present in almost all hyperthermophilic archaea whose whole genomes have been sequenced.

L-Lysine dehydrogenases catalyze the oxidative deamination of L-Lysine in the presence of NAD and so far two types of dehydrogenases were discovered. Thermophile and hyperthermophilic microorganisms were screened for a more stable form of L-Lysine dehydrogenase and results are discussed.

Researchers describe a dye-linked dehydrogenase from a thermophilic bacterium that reduces 2,6-dichloroindophenol in the presence of L-proline. This enzyme could be useful in biosensors and may be more stable than its counterpart in mesophilic bacteria.

Genes encoding from 2-deoxy-D-ribose-5-phosphate aldolases (DERAs) from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, are compared with Escherichia coli DERA in terms of their capacity to catalyze aldol condensation. The two enzymes from hyperthermophiles are highly resistant to acetaldehyde and have displayed greater catalysis of sequential aldol condensation of three acetaldehydes than the Escherichia coli enzyme, even at room temperature.

A study was made on the spore coat protein CotA from Bacillus subtilis, which was previously identified as a laccase. It was observed that the CotA showed a strong bilirubin oxidase activity and a remarkable affinity for bilirubin than conventional bilirubin oxidase.

The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned and functionally expressed in Escherichia coli. The deduced amino acid sequence showed a low level of identity to those of Escherichia coli ATP-dependant NAD kinase and the inorganic polyphosphate/ATP-dependent NAD kinase from a hyperthermophilic archaeon.

We recently identified and characterized a novel broad substrate specificity amino acid racemase (BAR) from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the first description of a PLP-dependent fold type I ASR in archaea. [ABSTRACT FROM AUTHOR]