title: Expanding the active pH range of Escherichia coli glutamate decarboxylase by breaking the cooperativeness.
abstract: Bacterial glutamate decarboxylase (GAD) transforms glutamate into γ-aminobutyric acid (GABA) with the consumption of a proton. The enzyme is active under acidic environments only and sharply loses its activity as pH approaches neutrality with concomitant structural deformation. In an attempt to understand better the role of this cooperative loss of activity upon pH shifts, we prepared and studied a series of GAD site-specific mutants. In this report, we show that the cooperativeness was kept intact by at least two residues, Glu89 and His465, of which Glu89 is newly identified to be involved in the cooperativity system of GAD. Double mutation on these residues not only broke the cooperativity in the activity change but also yielded a mutant GAD that retained the activity at neutral pH. The resulting mutant GAD that was active at neutral pH inhibited the cell growth in a glycerol medium by converting intracellular Glu into GABA in an uncontrolled manner, which explains in part why the cooperativeness of GAD has to be kept by several layers of safety keepers. This unexpected result might be utilized to convert a low-valued by-product of biodiesel production, glycerol, into value-added product, GABA.
authors.affiliation: Department of Chemical and Biochemical Engineering, Dongguk University-Seoul, Seoul 100-715, South Korea.
authors.forename: Ngoc Anh
authors.initials: NA
authors.lastname: Thu Ho
authors.forename: Chen Yuan
authors.initials: CY
authors.lastname: Hou
authors.forename: Woo Hyun
authors.initials: WH
authors.lastname: Kim
authors.forename: Taek Jin
authors.initials: TJ
authors.lastname: Kang
mesh_terms: D056804:Biofuels / Q000600:supply & distribution; D004926:Escherichia coli / Q000201:enzymology*; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D056804:Biofuels; D018698:Glutamic Acid; D006639:Histidine; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase