Gene Cloning and Characterization of the Enormous NAD-Dependent L-Glutamate Dehydrogenase from the psychrophile Janthinobacterium lividum isolated from cold soil

From the Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashiku, Fukuoka, 812-8581, and the Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima-shi, Tokushima, 770-8506

^* To whom correspondence should be addressed. Email: ohshima{at}agr.kyushu-u.ac.jp.

	Abstract

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 (1065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by SDS-PAGE). Consistent with those findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1575 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 enormous 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 those from S. clavuligerus and P. aeruginosa, but the central domains showed a 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 NADP, this GDH exclusively catalyzed the oxidative deamination of L-glutamate. The stereospecificity of the hydride transfer to NAD was proS, 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 (1735%) 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.