Purification and characterization of gamma-glutamylmethylamide synthetase from Methylophaga sp. AA-30

• Published in: Bioscience, biotechnology, and biochemistry Vol. 56; no. 5; pp. 708 - 711
• Main Authors: Kimura, T. (Mie Univ., Tsu (Japan). Faculty of Bioresources), Sugahara, I, Hanai, K, Tonomura, Y
• Format: Journal Article
• Language: English
• Published: Tokyo Taylor & Francis 1992; Japan Society for Bioscience Biotechnology and Agrochemistry
• Subjects: AMIDAS; AMIDE; AMIDES; BACTERIA; Biological and medical sciences; Biotechnology; Enzyme engineering; Fundamental and applied biological sciences. Psychology; Improved methods for extraction and purification of enzymes; LIGASAS; LIGASE; LIGASES; Methods. Procedures. Technologies; Methylophaga; PURIFICACION; PURIFICATION; Purification; γ-Glutamylmethylamide synthetase; Enzymatic activity; Enzyme; Substrate specificity; Isolation; Inhibition; Structure; Thermal stability
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.56.708

γ-Glutamylmethylamide synthetase [L-glutamate: methylamine ligase (ADP-forming), EC 6.3.4.12] was purified about 70-fold from a cell-free extract of Methylophaga sp. AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration. Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000. The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits. The enzyme had maximum activity at pH 7.5 and 40°C. It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.