Molecular analysis of two genes of the Escherichia coli gab cluster: nucleotide sequence of the glutamate:succinic semialdehyde transaminase gene (gabT) and characterization of the succinic semialdehyde dehydrogenase gene (gabD)

Abstract

We have characterized two genes of the Escherichia coli K-12 gab cluster, which encodes the enzymes of the 4-aminobutyrate degradation pathway. The nucleotide sequence of gabT, coding for glutamate:succinic semialdehyde transaminase (EC 2.6.1.19), alternatively known as 4-aminobutyrate transaminase, was determined. The structural gene consists of 1,281 nucleotides specifying a protein of 426 amino acids with a molecular mass of 45.76 kDa. The protein shows significant homologies to the ornithine transaminases from Saccharomyces cerevisiae and from rat and human mitochondria. Three functionally and structurally important amino acid residues of the transaminase were identified by sequence comparison studies, and evolutionary relationships of the aminotransferases are discussed. The gabD gene, encoding succinic semialdehyde dehydrogenase (EC 1.2.1.16), was cloned and shown to be located adjacent to the 5' end of gabT. Expression studies with subfragments of the initially cloned DNA region revealed a maximal size of 1.7 kb for gabD. Both genes are cotranscribed from a promoter located upstream of gabD.The gab cluster of Escherichia coli specifies the synthesis of the enzymes of the 4-aminobutyrate (GABA) degradation pathway (8,9). The cluster, which is located at 57.6 min on the E. coli K-12 chromosome, was mapped genetically by Metzer et al. (22) and shown to contain four genes: gabT, encoding glutamate: succinic semialdehyde transaminase (GSST; GABA transaminase; EC 2.6.1.19); gabD, encoding succinic semialdehyde dehydrogenase (SSDH; EC 1.2.1.16); gabP, encoding GABA permease; and a control gene, gabC, coordinately regulating their expression. In a previous work, we purified and characterized a GABA transaminase from E. coli K-12 (26). The corresponding gene was cloned on a 1.6-kb DraI-BamnHI fragment and overexpressed in E. coli, and its identity with gabT could be demonstrated (3). The gabT gene was shown to be situated on a 3.8-kb SalI-BamHI fragment together with the endogenous promoter (3). Moreover, we presented a restriction map of a 15-kb Sall fragment containing most of the E. coli gab cluster (3). Metzer and Halpern recently also reported the cloning of the E. coli K-12 gab region and suggested that the gab genes are divergently transcribed by two different promoters (21).In this paper, we report the complete nucleotide sequence of the GABA transaminase gene, gabT, and discuss the evolutionary relatedness of GABA transaminase to other aminotransferases. Furthermore, we describe the cloning and characterization of gabD and provide evidence that gabT and gabD are transcribed from a common promoter upstream of the gabD gene. MATERIALS AND METHODSBacterial strains, media, enzymes, and chemicals. For the transformation and expression experiments, the E. coli DH1 (17) and JM103 (20)

A Novel α-Ketoglutaric Semialdehyde Dehydrogenase

“…Plant SSALDH, for example, can utilize only succinic semialdehyde as a substrate but not propanal, glyceraldehyde, and glutaraldehyde, in contrast with ␣KGSA dehydrogenase (28). Furthermore, it is known that eukaryotic SSALDHs show NAD ϩ dependence (28,29), whereas bacterial ones show NADP ϩ dependence (30,31). Recently, two other members of this ALDH subfamily, toluenesulfonate aldehyde dehydrogenase (EC 1.2.1.62) (32) and 6-oxohexanoate dehydrogenase (EC 1.2.1.63) (33), were reported as NAD ϩ -dependent enzymes (Fig.…”

Activation of thegabOperon in an RpoS-Dependent Manner by Mutations That Truncate the Inner Core of Lipopolysaccharide inEscherichia coli

“…The gabT gene encodes a succinate:semialdehyde aminotransferase that catalyzes the formation of succinate semialdehyde from GABA. The gabP gene encodes a GABA permease (3). In addition to the catabolism of ␥-aminobutyrate, the gab operon has been proposed to contribute to polyamine homeostasis during nitrogen-limited growth (25) and to maintain high internal glutamate concentrations under stress conditions (14).…”

“…The gabDTPC operon in Escherichia coli functions in the conversion of ␥-aminobutyrate (GABA) to succinate (3,17). The gabD gene encodes a succinate:semialdehyde dehydrogenase that generates succinate from succinate-semialdehyde.…”

The RIB element in the goaG-pspF intergenic region of Escherichia coli

“…Enzymes with such activities are clustered in operons dedicated to the metabolism of arginine and ornithine in E. coli, such as the gabDTP cluster at 57 min and the speABC cluster at 66 min (6,33,38). A most puzzling observation is that the amino acid sequence of the activator of the 54 -dependent pspA-E operon, PspF, is more similar to E. coli ornithine decarboxylase inhibitor (Az) than to any other sequence in the data bank (27).…”