Structure of NADP+-dependent glutamate dehydrogenasefrom Escherichia coli – reflections on the basis ofcoenzyme specificity in the family of glutamatedehydrogenases

摘要

Glutamate dehydrogenases (GDHs; EC 1.4.1.2–4) catalyse the oxidativedeamination of L-glutamate to a-ketoglutarate, using NAD+ and/orNADP+ as a cofactor. Subunits of homo-hexameric bacterial enzymes comprisea substrate-binding domain I followed by a nucleotide-bindingdomain II. The reaction occurs in a catalytic cleft between the two domains.Although conserved residues in the nucleotide-binding domains of variousdehydrogenases have been linked to cofactor preferences, the structural basisfor specificity in the GDH family remains poorly understood. Here, therefined crystal structure of Escherichia coli GDH in the absence of reactantsis described at 2.5-A resolution. Modelling of NADP+ in domain II revealsthe potential contribution of positively charged residues from a neighbouringa-helical hairpin to phosphate recognition. In addition, a serine that followsthe P7 aspartate is presumed to form a hydrogen bond with the 2′-phosphate.Mutagenesis and kinetic analysis confirms the importance of these residuesin NADP+ recognition. Surprisingly, one of the positively charged residuesis conserved in all sequences of NAD+-dependent enzymes, but the conformationsadopted by the corresponding regions in proteins whose structurehas been solved preclude their contribution to the coordination of the2′-ribose phosphate of NADP+. These studies clarify the sequence–structurerelationships in bacterial GDHs, revealing that identical residues may specifydifferent coenzyme preferences, depending on the structural context. Primarysequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodateboth coenzymes in the dual-specificity GDHs of animals.