4BHT: Structural Determinants of Cofactor Specificity and Domain Flexibility in Bacterial Glutamate Dehydrogenases

Citation:
Abstract
Glutamate dehydrogenases (GDHs; EC 1.4.1.2–4) catalyse the oxidative deamination of l-glutamate to alpha-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 GDH family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5-A resolution. Modelling of NADP(+) in domain II reveals the potential contribution of positively charged residues from a neighbouring alpha-helical hairpin to phosphate recognition. In addition, a serine that follows the P7 aspartate is presumed to form a hydrogen bond with 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 to the coordination of the 2'-ribose phosphate of NADP(+) . These studies clarify the sequence-structure relationships in bacterial GDHs, 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.
PDB ID: 4BHTDownload
MMDB ID: 110992
PDB Deposition Date: 2013/4/6
Updated in MMDB: 2013/09 
Experimental Method:
x-ray diffraction
Resolution: 2.5  Å
Source Organism:
Similar Structures:
Biological Unit for 4BHT: hexameric; determined by author and by software (PISA)
Molecular Components in 4BHT
Label Count Molecule
Proteins (6 molecules)
6
Nadp-specific Glutamate Dehydrogenase(Gene symbol: gdhA)
Molecule annotation
Chemicals (20 molecules)
1
18
2
1
3
1
* Click molecule labels to explore molecular sequence information.

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