Location of the two genes encoding glutaconate coenzyme A-transferase at the beginning of the hydroxyglutarate operon in Acidaminococcus fermentans

Abstract

Glutaconate coenzyme A-transferase (Gct) from Acidaminococcus fermentans 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 encoding glutaconate CoA-transferase (gctAB) are located upstream of a gene cluster formed by gcdA, hgdC, hgdA and hgdB in this order. Further upstream of gctA, a DNA sequence was detected showing significant similarities to sigma 70-type promoters from Escherichia coli. Primer-extension analysis revealed that this specific DNA sequence was indeed the location of transcription initiation in A. fermentans. The entire gene cluster, 7.3 kb in length, comprising gctAB, gcdA and hgdCAB, has tentatively been named the hydroxyglutarate operon, since the enzymes encoded by these genes are involved in the conversion of (R)-2-hydroxyglutarate to crotonyl-CoA in the pathway of glutamate fermentation by A. fermentans. The genes gctAB were expressed together in E. coli. Cell-free extracts of a transformant E. coli strain contained glutaconate CoA-transferase at a specific activity of up to 30 U/mg protein. The recombinant enzyme was purified to homogeneity with a specific activity of 130 U/mg protein by ammonium sulfate fractionation and crystallisation. The amino acid residue directly involved in catalysis was tentatively identified as E54 of the small subunit of the enzyme (GctB).