PDBsum entry: 1k89

Oxidoreductase

PDB id: 1k89

Contents

Protein chain

449 a.a. *

Waters ×238

* Residue conservation analysis

PDB id:

1k89

Name:

Oxidoreductase

Title:

K89l mutant of glutamate dehydrogenase

Structure:

Glutamate dehydrogenase. Chain: a. Engineered: yes. Mutation: yes

Source:

Clostridium symbiosum. Organism_taxid: 1512. Gene: gdh. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Homo-Hexamer (from PDB file)

Resolution:

2.05Å R-factor: 0.143

Authors:

T.J.Stillman,A.M.B.Migueis,X.G.Wang,P.J.Baker,K.L.Britton,P. D.W.Rice

Key ref:

T.J.Stillman et al. (1999). Insights into the mechanism of domain closure and substrate specificity of glutamate dehydrogenase from Clostridium symbiosum. J Mol Biol, 285, 875-885. PubMed id: 9878450 DOI: 10.1006/jmbi.1998.2335

Date:

05-Jun-98 Release date: 27-Jan-99

Protein chain

P24295  (DHE2_CLOSY) -  NAD-specific glutamate dehydrogenase

Seq: 450 a.a.

Struc: 449 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.4.1.2 - Glutamate dehydrogenase.
• Reaction: L-glutamate + H₂O + NAD⁺ = 2-oxoglutarate + NH₃ + NADH

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: oxidation-reduction process (2 terms)
• Biochemical function: nucleotide binding (4 terms)

reference

DOI no: 10.1006/jmbi.1998.2335

J Mol Biol 285:875-885 (1999)

PubMed id: 9878450

Insights into the mechanism of domain closure and substrate specificity of glutamate dehydrogenase from Clostridium symbiosum.

T.J.Stillman, A.M.Migueis, X.G.Wang, P.J.Baker, K.L.Britton, P.C.Engel, D.W.Rice.

ABSTRACT

Comparisons of the structures of glutamate dehydrogenase (GluDH) and leucine dehydrogenase (LeuDH) have suggested that two substitutions, deep within the amino acid binding pockets of these homologous enzymes, from hydrophilic residues to hydrophobic ones are critical components of their differential substrate specificity. When one of these residues, K89, which hydrogen-bonds to the gamma-carboxyl group of the substrate l-glutamate in GluDH, was altered by site-directed mutagenesis to a leucine residue, the mutant enzyme showed increased substrate activity for methionine and norleucine but negligible activity with either glutamate or leucine. In order to understand the molecular basis of this shift in specificity we have determined the crystal structure of the K89L mutant of GluDH from Clostridium symbiosum. Analysis of the structure suggests that further subtle differences in the binding pocket prevent the mutant from using a branched hydrophobic substrate but permit the straight-chain amino acids to be used as substrates.The three-dimensional crystal structure of the GluDH from C. symbiosum has been previously determined in two distinct forms in the presence and absence of its substrate glutamate. A comparison of these two structures has revealed that the enzyme can adopt different conformations by flexing about the cleft between its two domains, providing a motion which is critical for orienting the partners involved in the hydride transfer reaction. It has previously been proposed that this conformational change is triggered by substrate binding. However, analysis of the K89L mutant shows that it adopts an almost identical conformation with that of the wild-type enzyme in the presence of substrate. Comparison of the mutant structure with both the wild-type open and closed forms has enabled us to separate conformational changes associated with substrate binding and domain motion and suggests that the domain closure may well be a property of the wild-type enzyme even in the absence of substrate.

Selected figure(s)

Figure 1.
Figure 1. (a) A stereoview of a superposition of the active site residues in the closed form of GluDH (black) with those of the K89L mutant (grey) produced by the MIDAS display program [Ferrin et al 1988]. The glutamate substrate of GluDH is shown (Glu 501) together with the mutated residue (K89L), and atoms within hydrogen-bonding distances are linked with broken lines. (b), (c) and (d) Three stereo representations of the electron density in the active sites of the K89L mutant and the closed and open forms of wild-type GluDH, respectively, produced by the program BOBSCRIPT [Esnouf 1997]. The electron density associated with the substrate can be clearly seen in (c).

Figure 4.
Figure 4. A stereoview (MIDAS) of a superposition of the active-site residues of both the open (white) and closed (black) forms of GluDH. The main movements within the active site on domain closure are to residues Lys113, Val377, Leu378 and Ser380.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1999, 285, 875-885) copyright 1999.

Figures were selected by an automated process.