Isolation, Overexpression, and Biochemical Characterization of the Two Isoforms of Glutamic Acid Decarboxylase fromEscherichia coli

Abstract

Escherichia coliglutamic acid decarboxylase is a pyridoxal phosphate-dependent enzyme that catalyzes the α-decarboxylation of glutamate to yield 4-aminobutyrate and CO2. TheE. colichromosome contains two genes encoding for this enzyme,gadAandgadB,which map at distinctloci.Their protein products differ in only five amino acid residues, four of which are located in the N-terminal region (Smithet al.,1992,J. Bacteriol.174, 5820–5826). Herein, we report the sequences of the twogadgenes, including their regulatory regions. Both genes were separately cloned into the vector pQE60, for overexpression under the control of thelacpromoter. In this way, we have succeded in separately expressing large quantities of each pure isoform. The two isoforms were characterized biochemically and all evidence, including that from analysis of the complex pre-steady-state kinetic behavior of the enzymes, indicates that the functional properties of the two isoenzymes are identical.