The crystal structure of glutamate dehydrogenase from Clostridium symbiosum at 0.6 nm resolution

Abstract

The structure of glutamate dehydrogenase from Clostridium symbiosum has been solved by single-crystal X-ray-diffraction studies at 0.6 nm resolution by using a combination of isomorphous replacement and molecular averaging. The electron-density map reveals that this glutamate dehydrogenase is a hexameric oligomer, arranged in 32 symmetry, of cylindrical appearance and dimensions, of length 10.8 nm and radius 4.4 nm. From an analysis of this map each subunit appears to contain some 55% alpha-helix and is organized into two distinct globular domains separated by a deep cleft. The subunits associate using the domain closest to the 32-symmetry point, making intimate contacts around the threefold and twofold interfaces. The second domain shows structural homology to the NAD-binding domain of other dehydrogenases, and difference Fourier analysis has shown that the NAD is bound in both a structurally equivalent position and a similar conformation to that observed for those related enzymes.

Alteration in relative activities of phenylalanine dehydrogenase towards different substrates by site‐directed mutagenesis

“…In seeking to decipher the rules governing the activity of these enzymes with different substrates, it is now possible to compare their amino acid sequences in the light of the known structure of glutamate dehydrogenase (GDH) from Clostridium ~ymbiosum [2,[9][10][11]. Though the sequences of both PheDH and LeuDH show a relatively low level of sequence identity with GDH, the alignment suggests that residues inferred as being responsible for the overall shape of the active site and for catalysis, as well as those involved in the recognition of the nicotinamide cofactor, are conserved in all three enzymes *Corresponding author.…”

Identification of the GTP Binding Site of Human Glutamate Dehydrogenase by Cassette Mutagenesis and Photoaffinity Labeling

“…GDH (EC 1.4.1.2-1.4.1.4) catalyzes the reversible deamination of L-glutamate to 2-oxoglutarate using NAD ϩ or NADP ϩ as coenzyme (1). The largest difference between mammalian and bacterial GDH is a long antenna domain formed by the 48-amino acid insertion extending from the top of the NAD domain and lying adjacent to the 3-fold axis of the hexamer (2)(3)(4)(5), and there is little identity between the 100 residues in the C terminus (6). In contrast to the extensive allosteric homotrophic and heterotrophic regulation observed in mammalian GDH, bacterial forms of GDH are relatively unregulated.…”

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance

“…While RocG is an enzymatically active GDH, GudB is inactive, due to a duplication of three amino acid residues at its active center. Decryptification of gudB in a rocG background is achieved by high-frequency acquisition of a suppressor mutation consisting of the precise deletion of part of the 9-bp direct repeat that prevents activity [46,47]. Both RocG and decryptified GudB are primarily catabolic dehydrogenases, and de novo glutamate synthesis in B. subtilis is performed exclusively by GOGAT.…”