Structure of NADP⁺‐dependent glutamate dehydrogenase from Escherichia coli – reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases

Abstract

Summary Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of l-glutamate to α-ketoglutarate using NAD+ and/or NADP+ as a cofactor. Subunits of homo-hexameric bacterial enzymes comprise a substrate-binding Domain I followed by a nucleotide binding Domain II. The reaction occurs in a catalytic cleft between the two domains. Although conserved residues in the nucleotide-binding domains of various dehydrogenases have been linked to cofactor preferences, the structural basis for specificity in the glutamate dehydrogenase (GDH) family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5Å resolution. Modelling of NADP+ in Domain II reveals the potential contribution of positively charged residues from a neighbouring α-helical hairpin to phosphate recognition. In addition, a serine residue that follows the P7 aspartate is presumed to form a hydrogen bond to the 2’-phosphate. Mutagenesis and kinetic analysis confirms the importance of these residues in NADP+ recognition. Surprisingly, one of the positively charged residues is conserved in all sequences of NAD+ dependent enzymes, but the conformations adopted by the corresponding regions in proteins whose structure has been solved preclude their contribution toward the co-ordination of the 2’-ribose phosphate of NADP+. These studies clarify the sequence/structure relationships in bacterial glutamate dehydrogenases, revealing that identical residues may specify different coenzyme preferences, depending on the structural context. Primary sequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodate both coenzymes in the dual specificity GDHs of animals.

Structural basis for the catalytic mechanism and α-ketoglutarate cooperativity of glutamate dehydrogenase

“…S2) with other NADP + /NAD + -specific GDHs and a few extra amino acid insertions. The overall structural fold of AnGDH is similar to the previously determined structures of E. coli GDH (EcGDH) and other GDHs (7,12,(21)(22)(23). Each subunit of AnGDH consists of two domains separated by a deep cleft (Fig.…”

“…The NADP + -specific bacterial/fungal GDHs and the dual coenzyme specific mammalian GDHs are hexameric (12). The NAD + -dependent bacterial/fungal enzymes are either homohexamers (13) or homo-tetramers (14,15).…”

“…Despite numerous studies in the last 40 years to identify the intermediates formed during the reaction catalyzed by GDHs (16)(17)(18)(19)(20), structural basis of the reaction mechanism of this enzyme remains unresolved. A number of medium/low resolution crystal structures of GDHs have been determined as complexes with substrates (α-ketoglutarate or glutamate) and coenzymes (NADP + or NAD + ) (7,12,13,16,(21)(22)(23). However, these structures fail to explain the reaction mechanism and the role of different active site residues in catalysis, mainly due to their limited resolution and unavailability of any intermediate-bound, catalytically competent structures of this enzyme.…”

NADP + -Preferring d -Lactate Dehydrogenase from Sporolactobacillus inulinus

“…Unlike D-LDHs, which are all reported to be NAD ϩ dependent to date, three types of GDHs have been identified: NAD ϩ dependent, NADP ϩ dependent, and dual-nucleotide specific (32). As DLDH744 could use both NADH and NADPH with high efficiencies and even with a preference for NADPH as the coenzyme both in vitro and in vivo, protein structures were first compared for DLDH744 and GDHs.…”

“…Consequentially, the focus of the study was shifted to investigating the difference between DLDH744 and other reported NAD-linked LDHs with experimentally verified functionalities. It was reported that primary sequence alone is not a reliable guide for predicting coenzyme specificity (32). Cofactor specificity appears to be a function of the arrangement of secondary structural elements.…”