Selection of Escherichia coli Glutamate Decarboxylase Active at Neutral pH from a Focused Library

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Bacterial glutamate decarboxylase (GAD) converts glutamate (Glu) into -aminobutyric acid (GABA) at acidic conditions. Since the reaction consumes a proton per GABA synthesis, cells use this reaction to survive in the acidic environments. Characteristically, the enzyme displays a sigmoidal decrease in its activity as pH rises becoming completely inactive at or above pH 6. This cooperative activity loss is accompanied by several distinct structural changes. Previously, by examining structures at acidic and neutral pH, two key regions had been chosen and mutated to break the cooperativity; Glu89 and C-terminal 15 residues. In this study, we included Asp86 in candidate key residues for mutation to break the cooperativity of GAD. We devised a selection strategy according to which only Escherichia coli cells expressing a variant GAD that was active at neutral pH could survive. In this scheme, an alanine (Ala) auxotroph was rescued by the intracellular synthesis of GABA that was subsequently converted into Ala by heterologously expressed GABA-pyruvate transaminase. New GAD variants were readily selected using this strategy and the most of them indeed had a mutation at residue 86. The results suggest that the role of Asp86 in the wild-type enzyme might be the same as Glu89; to make GAD keep its activity only at acidic environments. Characterization of representative variants are also presented.
Publisher
KOREAN SOC BIOTECHNOLOGY & BIOENGINEERING
Issue Date
2018-08
Language
English
Article Type
Article
Keywords

GAMMA-AMINOBUTYRIC-ACID; GABA SHUNT; RANGE; FOODS

Citation

BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, v.23, no.4, pp.473 - 479

ISSN
1226-8372
DOI
10.1007/s12257-018-0258-9
URI
http://hdl.handle.net/10203/245915
Appears in Collection
BS-Journal Papers(저널논문)
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