Gene cloning and characterization of the very large NAD-dependent l-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil

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 (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.

FIG. 1.

PAGE of purified GDH from J. lividum UTB1302. (A) Native PAGE on a 7.5% acrylamide gel was performed at 4°C. The left lane shows active staining, and the right lane shows protein staining. The arrows indicate the position of the native protein. (B) SDS-PAGE on a 10% acrylamide gel.

FIG. 2.

Nucleotide and deduced amino acid sequences of J. lividum GDH. The N-terminal amino acid sequence determined by protein sequencing is underlined. The full nucleotide sequence was obtained by sequencing pgdh1, pgdh2, and pgdh3, which were comprised of genomic SphI (nucleotide positions 1 to 2295), SalI (nucleotide positions 2243 to 2821), and SphI (nucleotide positions 2296 to 5098) fragments, respectively. The amino acid sequence of the central domain (amino acids 719 to 1190) is shaded.

FIG. 3.

SDS-PAGE of recombinant GDH purified from E. coli. Lane 1, molecular markers; lane 2, crude extract; lane 3, Butyl Toyopearl fraction; lane 4, Superdex 200 fraction.

FIG. 4.

Saturation curve for enzyme activity elicited by addition of l-aspartate.

FIG. 5.

Portion of the ¹H NMR spectra for NADH produced by the enzyme reaction with l-glutamate (A) and dl-glutamate (2,4,4-d₃) (B).

FIG. 6.

Amino acid sequence alignment of the central domains of P. aeruginosa, S. clavuligerus, and J. lividum GDHs (170 to 180 kDa) and C. symbiosum GDH-50. Sequences were aligned using Clustal W (24). Residues conserved in the three very large GDHs and in all the sequences are indicated by shading and asterisks, respectively. Residues that interact with l-glutamate and that are involved with the catalytic reaction are indicated by filled circles. Residues responsible for the pro-S specificity of the hydrogen transfer to NADH are enclosed in a box.