NADP+âdependent glutamate dehydrogenase in the Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1: Characterization, protein and DNA sequence, and relationship to other glutamate dehydrogenases
2000
Di Fraia, Raffaela | Wilquet, Valerie | Ciardiello, M Antonietta | Carratore, Vito | Antignani, Antonella | Camardella, Laura | Glansdorff, Nicolas | di Prisco, Guido
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+âdependent GDH has been purified to homogeneity and the gene encoding GDH has been cloned and expressed. The enzyme has a hexameric structure. The amino acid sequence determined by peptide and gene analyses comprises 447 residues, yielding a protein with a molecular mass of 49â285âDa. The sequence shows homology with hexameric GDHs, with identity levels of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respectively. The coenzymeâbinding fingerprint motif GXGXXG/A (common to all GDHs) has Ser at the last position in this enzyme. The overall hydrophilic character is increased and a fiveâresidue insertion in a loop between two αâhelices may contribute to the increase in protein flexibility. Psychrobacter sp. TAD1 GDH apparent temperature optimum is shifted towards low temperatures, whereas irreversible heat inactivation occurs at temperatures similar to those of E.âcoli GDH. The catalytic efficiency in the temperature range 10–30â°C is similar or lower than that of E.âcoli GDH. Unlike E.âcoli GDH the enzyme exhibits marked positive cooperativity towards 2âoxoglutarate and NADPH. This feature is generally absent in prokaryotic GDHs. These observations suggest a regulatory role for this GDH, the most crucial feature being the structural/functional properties required for fine regulation of activity, rather than the high catalytic efficiency and thermolability encountered in several coldâactive enzymes.
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