PDBsum entry: 1b3b

Oxidoreductase

PDB id: 1b3b

Contents

Protein chains

(+ 0 more) 409 a.a. *

* Residue conservation analysis

PDB id:

1b3b

Name:

Oxidoreductase

Title:

Thermotoga maritima glutamate dehydrogenase mutant n97d, g376k

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:

3.10Å R-factor: 0.225 R-free: 0.298

Authors:

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

Key ref:

J.H.Lebbink et al. (1998). Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. I. Introduction of a six-residue ion-pair network in the hinge region. J Mol Biol, 280, 287-296. PubMed id: 9654452 DOI: 10.1006/jmbi.1998.1870

Date:

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

Protein chains

P96110  (DHE3_THEMA) -  Glutamate dehydrogenase

Seq: 416 a.a.

Struc: 409 a.a.*

* PDB and UniProt seqs differ at 3 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.1998.1870

J Mol Biol 280:287-296 (1998)

PubMed id: 9654452

Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. I. Introduction of a six-residue ion-pair network in the hinge region.

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

ABSTRACT

Comparison of the recently determined three-dimensional structures of several glutamate dehydrogenases allowed for the identification of a five-residue ion-pair network in the hinge region of Pyrococcus furiosus glutamate dehydrogenase (melting temperature 113 degrees C), that is not present in the homologous glutamate dehydrogenase from Thermotoga maritima (melting temperature 93 degrees C). In order to study the role of this ion-pair network, we introduced it into the T. maritima enzyme using a site-directed mutagenesis approach. The resulting T. maritima glutamate dehydrogenases N97D, G376 K and N97D/G376 K as well as the wild-type enzyme were overproduced in Escherichia coli and subsequently purified. Elucidation of the three-dimensional structure of the double mutant N97D/G376 K at 3.0 A, showed that the designed ion-pair interactions were indeed formed. Moreover, because of interactions with an additional charged residue, a six-residue network is present in this double mutant. Melting temperatures of the mutant enzymes N97D, G376 K and N97D/G376 K, as determined by differential scanning calorimetry, did not differ significantly from that of the wild-type enzyme. Identical transition midpoints in guanidinium chloride-induced denaturation experiments were found for the wild-type and all mutant enzymes. Thermal inactivation at 85 degrees C occured more than twofold faster for all mutant enzymes than for the wild-type glutamate dehydrogenase. At temperatures of 65 degrees C and higher, the wild-type and the three mutant enzymes showed identical specific activities. However, at 58 degrees C the specific activity of N97D/G376 K and G376 K was found to be significantly higher than that of the wild-type and N97D enzymes. These results suggest that the engineered ion-pair interactions in the hinge region do not affect the stability towards temperature or guanidinium chloride-induced denaturation but rather affect the specific activity of the enzyme and the temperature at which it functions optimally.

Selected figure(s)

Figure 2.
Figure 2. Schematic drawing of the hinge ion-pair network in wild-type P. furiosus GDH (a), wild-type T. maritima GDH (b) and in double mutant N97D/G376 K (c). The orientation of the secondary structure elements is similar to that in Figure 1. The green-coloured loop and # character indicate residues from an adjacent subunit. The Figure has been generated with the programme MOLSCRIPT [Kraulis 1991] and the rendering programme Raster3D [Bacon and Anderson 1988 and Merrit and Murphy 1994].

Figure 3.
Figure 3. (a) 6-fold averaged (F[O]^Mu−F[C]^WT) exp(iα[C]^WT)map (difference map) of double mutant N97D/G376 K. Shown are electron densities at residues 376, 97 and 64. The map has been contoured at 1.5σ. (b) Averaged density map (2F[O]^MU−F[C]^MU) exp(iα[C]^MU) of five residues involved in the network, contoured at 1.5 σ.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1998, 280, 287-296) copyright 1998.

Figures were selected by an automated process.