NADP(+)-dependent glutamate dehydrogenase in the Antarctic psychrotolerantbacterium Psychrobacter sp TAD1 - Characterization, protein and DNA sequence, and relationship to other glutamate dehydrogenases

The Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1 contains two distinct glutamate dehydrogenases (GDH), each specific for either NADP(+) or NAD(+). This feature is quite unusual in bacteria, which generally have a single GDH. NADP(+)-dependent GDH has been purified to homogeneity and th e gene encoding GDH has been cloned and expressed. The enzyme has a hexamer ic structure. The amino acid sequence determined by peptide and gene analys es comprises 447 residues, yielding a protein with a molecular mass of 49 2 85 Da. The sequence shows homology with hexameric GDHs, with identity level s of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respe ctively. The coenzyme-binding fingerprint motif GXGXXG/A (common to all GDH s) has Ser at the last position in this enzyme. The overall hydrophilic cha racter is increased and a five-residue insertion in a loop between two alph a-helices may contribute to the increase in protein flexibility. Psychrobac ter sp. TAD1 GDH apparent temperature optimum is shifted towards low temper atures, whereas irreversible heat inactivation occurs at temperatures simil ar to those of E. coli GDH. The catalytic efficiency in the temperature ran ge 10-30 degrees C is similar or lower than that of E. coli GDH. Unlike E. coli GDH the enzyme exhibits marked positive cooperativity towards 2-oxoglu tarate and NADPH. This feature is generally absent in prokaryotic GDHs. The se observations suggest a regulatory role for this GDH, the most crucial fe ature being the structural/functional properties required for fine regulati on of activity, rather than the high catalytic efficiency and thermolabilit y encountered in several cold-active enzymes.