Overexpression and site-directed mutagenesis of the succinyl-CoA synthetase of Escherichia coli and nucleotide sequence of a gene (g30) that is adjacent to the suc operon

Abstract

The succinyl-CoA synthetase of Escherichia coli is encoded by two genes, sucC (beta subunit) and sucD (alpha subunit), which are distal genes in the sucABCD operon. They are expressed from the suc promoter, which also expresses the dehydrogenase and dihydrolipoyl succinyl-transferase subunits of the 2-oxoglutarate dehydrogenase complex. Strategies have now been devised for the site-directed mutagenesis and independent expression of the succinyl-CoA synthetase (alpha 2 beta 2 tetramer) and the individual subunits. These involve (1) subcloning a promoterless sucCD fragment downstream of the lac promoter in M13mp10, and (2) precise splicing of the suc coding regions with the efficient atpE ribosome-binding site and expression from the thermoinducible lambda promoters in the pJLA503 vector. Succinyl-CoA synthetase specific activities were amplified 40-60-fold within 5 h of thermoinduction of the lambda promoters, and the alpha and beta subunits accounted for almost 30% of the protein in supernatant fractions of the cell-free extracts. Site-directed mutagenesis of potential CoA binding-site residues indicated that Trp-43 beta and His-50 beta are essential residues in the beta-subunit, whereas Cys-47 beta could be replaced by serine without inactivating the enzyme. No activity was detected after the histidine residue at the phosphorylation site of the alpha-subunit was replaced by aspartate (His-246 alpha----Asp), but this alteration seemed to have a deleterious effect on the accumulation of the enzyme in cell-free supernatant extracts. The nucleotide sequence of an unidentified gene (g30) that is adjacent to the sucABCD operon was defined by extending the sequence of the citric acid cycle gene cluster by 818 bp to 13379 bp: gltA-sdhCDAB-sucABCD-g30. This gene converges on the suc operon and encodes a product (P30) that contains 230 amino acids (Mr 27,251). Highly significant similarities were detected between the N-terminal region of P30 and those of GENA [the product of another unidentified gene (geneA) located upstream of the aceEF-lpd operon], and GNTR (a putative transcriptional repressor of the gluconate operon of Bacillus subtilis). Possible roles for GENA and P30 as transcriptional regulators of the adjacent operons encoding the pyruvate and 2-oxoglutarate dehydrogenase complexes are discussed.

Transfer functions of the conjugative integrating element pSAM2 from Streptomyces ambofaciens: characterization of a kil-kor system associated with transfer

“…5a). Interestingly, pIJ101 Tra protein may also be a nucleotide-binding protein NruI (8) (28). However, it should be noted that traSA of pSAM2 is predicted to encode a protein half the size (306 aa) of that encoded by tra of pIJ101 (621 aa).…”

“…This family includes the E. coli P30 protein (240 aa) (8), which is possibly a transcriptional regulator of the succinyl coenzyme A synthetase operon (22.8% identity with KorSA in a 240-aa overlap); the E. coli A protein (236 aa) (50) (22.0% identity in a 123-aa overlap), which is a possible transcriptional regulator for the pyruvate dehydrogenase complex; and the histidine utilization repressors (HUTC) of Pseudomonas putida (1) (21).…”

Novel phosphotransferase system genes revealed by bacterial genome analysis - a gene cluster encoding a unique Enzyme I and the proteins of a fructose-like permease system

“…We have recently sequenced a 4.5 kbp region of the Salmonella chromosome between y s M and crr and have identified an ORF which encodes a protein that possesses an identifiab!e N-terminal helix-turn-helix motif homologous to those of members of the B. subtilis gluconate repressor (GntR) family (Titgemeyer etal., 1994;Buck & Guest, 1989;Reizer et al, 1991a). N o ORF encoding an Enzyme I-like protein was found in this region.…”

icdB mutants of Escherichia coli

“…Enzyme assays showed that icdB mutants lysogenized by 176 were proficient for citrate synthase. The cloned E. coli DNA in 176 includes gltA, sdhCDAB (succinate dehydrogenase), sucAB (2-oxoglutarate dehydrogenase), sucCD (succinyl-coenzyme A synthetase), and orfg30 (possibly a regulatory gene) (2,10). 177 contains sucCD, orfg30, and DNA extending clockwise from orfg30.…”