title: C-terminal truncation of glutamate decarboxylase from Lactobacillus brevis CGMCC 1306 extends its activity toward near-neutral pH.
abstract: Glutamate decarboxylase (GAD) from Lactobacillus brevis is a very promising candidate for biosynthesis of GABA and various other bulk chemicals that can be derived from GABA. However, no structure of GAD of this origin has been reported to date, which limits enzyme engineering strategy to improve its properties for better use in production of GABA. Bacterial GAD exhibits an acidic pH optimum and there is often a sharp pH dependence. In the present work, site-directed mutagenesis was performed to delete the C-terminal residues of GAD to generate a mutant, designated as GADΔC, which exhibited extended activity toward near-neutral pH compared to the wild type. Comparison of the UV-visible, fluorescence and Circular Dichroism spectra of the mutant with those of the wild type revealed that the microenvironment of the active site had been changed. Based on the homology model, we speculated that the substrate entrance was probably enlarged in GADΔC. These results provide evidence for the important role of C-terminal region in the pH-dependent regulation of enzyme activity, and the resulting mutant would be useful in a bioreactor for continuous production of GABA.
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Yu
authors.forename: Ling
authors.initials: L
authors.lastname: Lin
authors.forename: Sheng
authors.initials: S
authors.lastname: Hu
authors.forename: Jun
authors.initials: J
authors.lastname: Huang
authors.forename: Lehe
authors.initials: L
authors.lastname: Mei
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001709:Biotechnology / Q000379:methods; D020134:Catalytic Domain; D002942:Circular Dichroism; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D052196:Levilactobacillus brevis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase