Molecular and General Genetics MGG

, Volume 257, Issue 3, pp 338–347

Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs

  • R. N. Z. A. Rahman
  • S. Fujiwara
  • M. Takagi
  • T. Imanaka
ORIGINAL PAPER

DOI: 10.1007/s004380050655

Cite this article as:
Rahman, R., Fujiwara, S., Takagi, M. et al. Mol Gen Genet (1998) 257: 338. doi:10.1007/s004380050655

Abstract

The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25 GDH sequences including KOD1-GDH, and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both enzymes had a molecular mass of 47 300 Da and were shown to be functional in a hexameric form (284 kDa). The N-terminal amino acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar to those of the native GDH, except for a lower level of thermostability, with a half-life of 2 h at 100° C, compared to 4 h for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production. KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs.

Key words Glutamate dehydrogenaseArchaeaGene expressionThermostabilityAmmonia assimilation

Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • R. N. Z. A. Rahman
    • 1
  • S. Fujiwara
    • 2
  • M. Takagi
    • 2
  • T. Imanaka
    • 1
  1. 1.Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-01, Japan Fax: +81-075-753-4703JP
  2. 2.Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, JapanJP