Characterization of recombinant glutamine synthetase from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

Applied and Environmental Microbiology
R N Adul RahmanT Imanaka

Abstract

The glnA gene encoding glutamine synthetase was cloned from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1, and its nucleotide sequence was determined. The glnA gene was expressed in Escherichia coli ME8459 (glnA mutant strain), and the protein was purified to homogeneity and shown to be functional in a dodecameric from (637,000 Da), exhibiting both transferase and synthetase activities. However, kinetic studies indicated that the enzyme possessed low biosynthetic activity, suggesting that the reaction was biased towards glutamate production. The optimum temperature for both activities was 60 degrees C, which was lower than the optimal growth temperature of KOD1. Recombinant KOD1 GlnA exhibited different optimum pHs depending on the reaction employed (pH 7.8 for the synthetase reaction and pH 7.2 for the transferase reaction). Of the various nucleoside triphosphates tested, GTP as well as ATP was involved in the synthetase reaction.

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Citations

Nov 6, 2004·Microbiology·Purificación CabelloConrado Moreno-Vivián
Dec 12, 2012·Extremophiles : Life Under Extreme Conditions·Kenta TagashiraTadayuki Imanaka
Sep 26, 2001·Applied and Environmental Microbiology·P RobinsonM F Roberts
Oct 29, 2000·Journal of Bacteriology·M NakataniT Imanaka
Dec 8, 2006·Molecular & Cellular Proteomics : MCP·Na SunIstván Nagy
Oct 6, 2006·FEMS Microbiology Letters·Rosa María Martínez-EspinosaMaría José Bonete
Mar 10, 2001·Biochemical and Biophysical Research Communications·X F TangT Imanaka
Nov 15, 1997·Applied and Environmental Microbiology·M TakagiT Imanaka
Oct 26, 2010·Water Research·Octavio Perez-GarciaYoav Bashan

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