PDBsum entry: 2tmg

Oxidoreductase

PDB id: 2tmg

Contents

Protein chains

(+ 0 more) 408 a.a. *

* Residue conservation analysis

PDB id:

2tmg

Name:

Oxidoreductase

Title:

Thermotoga maritima glutamate dehydrogenase mutant s128r, t158e, n117r, s160e

Structure:

Protein (glutamate dehydrogenase). Chain: a, b, c, d, e, f. Engineered: yes. Mutation: yes

Source:

Thermotoga maritima. Organism_taxid: 2336. Cellular_location: cytoplasm. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Hexamer (from PQS)

Resolution:

2.90Å R-factor: 0.211 R-free: 0.274

Authors:

S.Knapp,J.H.G.Lebbink,J.Van Der Oost,W.M.De Vos,D.Rice, R.Ladenstein

Key ref:

J.H.Lebbink et al. (1999). Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface. J Mol Biol, 289, 357-369. PubMed id: 10366510 DOI: 10.1006/jmbi.1999.2779

Date:

04-Dec-98 Release date: 08-Dec-99

Protein chains

P96110  (DHE3_THEMA) -  Glutamate dehydrogenase

Seq: 416 a.a.

Struc: 408 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.4.1.3 - Glutamate dehydrogenase (NAD(P)(+)).
• Reaction: L-glutamate + H₂O + NAD(P)(+) = 2-oxoglutarate + NH₃ + NAD(P)H

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.1999.2779

J Mol Biol 289:357-369 (1999)

PubMed id: 10366510

Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface.

J.H.Lebbink, S.Knapp, J.van der Oost, D.Rice, R.Ladenstein, W.M.de Vos.

ABSTRACT

The role of an 18-residue ion-pair network, that is present in the glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been studied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. These two GDHs are 55 % identical in amino acid sequence, differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T. maritima glutamate dehydrogenase showed that all anticipated ion-pairs are formed and that a 16-residue ion-pair network is present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilizing single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85 degrees C, a 3 degrees C higher temperature optimum for catalysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis that large ion-pair networks do indeed stabilize enzymes from hyperthermophilic organisms.

Selected figure(s)

Figure 2.
Figure 2. Schematic presentation of the 18-residue ion-pair network in P. furiosus GDH (a) and the homologous residues in T. maritima GDH (b). Residues forming ion-pairs are connected by dotted lines, broken lines indicate subunit interfaces.

Figure 4.
Figure 4. Stereoview of the 16-residue network in T. maritima quadruple mutant GDH.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1999, 289, 357-369) copyright 1999.

Figures were selected by an automated process.