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).

Meta-cleavage enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni TA441

“…The deduced N-terminal amino acid sequence of the ORF2 product (SEIALVDRVICAAA-RAWENDGEVLATGIGL) was almost identical to that of TIP6 (SEIALVDRVIEAAARAWENDGEVLATGI-GL ; 29 of 30 amino acid residues were the same), one of the testosterone-inducible proteins in C. testosteroni ATCC 11996 (Mo$ bus et al, 1997). The deduced amino acid sequences of ORF1 and ORF2 showed 26n9% identity with subunit A of glutaconate CoA-transferase, which is involved in glutamate metabolism in Acidaminococcus fermentans, and 22n4 % identity with subunit B, respectively (Mack et al, 1994 ;Jacob et al, 1997). Glutaconate CoA-transferase produces acetate and glutaconyl-1-CoA from acetyl-CoA and (E)-glutaconate.…”

Unusual DAhydrations in anaerobic bacteria: considering ketyls (radical anions) as reactive intermediates in enzymatic reactions

“…A final CoA transfer leads to the free fatty acid, which is excreted. The most intensive studied pathway of this type is the fermentation of glutamate via (R)-2-hydroxyglutarate to ammonia, carbon dioxide, acetate, butyrate and molecular hydrogen by the mesophilic anaerobe Acidaminocoecus fermentans isolated from the intestine of mammals [6,7]. Glutamate is initially oxidized to 2-oxoglutarate and then reduced to (R)-2-hydroxyglutarate.…”

“…The genes from A. fermentans coding for the three polypeptides (a,[3 and 7) are clustered together in All rights reserved. the order hgdCAB, which form the 3'-end of the 'hydroxyglutarate operon', comprising in addition gctA and B coding for glutaconate CoA-tranferase as well as gcdA coding for the hydrophilic carboxytransferase subunit of the biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase, the consecutive enzyme of this pathway [7,17]. The extremely oxygen-sensitive activator HgdC (half-life under air at 25°C approx.…”

An Asymmetric Model for Na+-translocating Glutaconyl-CoA Decarboxylases

“…Hence gcdA is located upstream of hgdCAB as already observed in A. fermentans and F. nucleatum. Similarly, upstream of gcdA the two gctAB genes coding for glutaconate CoA-transferase were detected (9). The sequence of the DNA fragment comprising gcdA was deposited in GenBank TM under accession number FJ390113.…”

“…The gcdA gene coding for the carboxytransferase (GcdA) is part of the 2-hydroxyglutarate operon from A. fermentans (8,9). The three other genes, gcdDCB, form a separate transcription unit, preceded by mcdA, encoding the putative carboxyltransferase subunit of methylmalonyl-CoA decarboxylase ( Fig.…”