LOCATION OF THE 2 GENES ENCODING GLUTACONATE COENZYME-A-TRANSFERASE AT THE BEGINNING OF THE HYDROXYGLUTARATE OPERON IN ACIDAMINOCOCCUS-FERMENTANS

Glutaconate coenzyme A-transferase (Get) from Acidaminococcus fermenta ns consists of two subunits (GctA, 35725 Da and GctB, 29168 Da). The N -termini sequences of both subunits were determined. DNA sequencing of a subgenomic fragment of A. fermentans revealed that the genes encodi ng glutaconate CoA-transferase (gctAB) are located upstream of a gene cluster formed by gcdA, hgdC, hgdA and hgdB in this order. Further ups tream of gctA, a DNA sequence was detected showing significant similar ities to sigma(70)-type promoters from Escherichia coli. Primer-extens ion analysis revealed that this specific DNA sequence was indeed the l ocation of transcription initiation in A. fermentans. The entire gene cluster, 7.3 kb in length, comprising gctAB, gcdA and hgdCAB, has tent atively been named the hydroxyglutarate operon, since the enzymes enco ded by these genes are involved in the conversion of (R)-2-hydroxyglut arate to crotonyl-CoA in the pathway of glutamate fermentation by A. f ermentans. The genes gctAB were expressed together in E. coli. Cell-fr ee extracts of a transformant E. coli strain contained glutaconate CoA -transferase at a specific activity of up to 30 U/mg protein. The reco mbinant enzyme was purified to homogeneity with a specific activity of 130 U/mg protein by ammonium sulfate fractionation and crystallisatio n. The amino acid residue directly involved in catalysis was tentative ly identified as E54 of the small subunit of the enzyme (GctB).