Cloning and sequencing of the genes of 2‐hydoxyglutaryl‐CoA dehydratase from Acidaminococcus fermentans

Abstract

Two genomic libraries from Acidaminococcus fermentans DNA constructed with the λ vectors gt11 and EMBL 3 were screened with antisera raised against 2‐hydroxyglutaryl‐CoA dehydratase. Two clones giving the strongest reaction in the immunoassay were analyzed further, one was a λgt11 clone with an insert of 2050 bp and one was a λEMBL‐3 clone with an insert of approximately 11000 bp. Escherichia coli cells infected with the λgt11 clone expressed the α subunit of the dehydratase (M_r, 53870), whereas with the λEMBL‐3 clone, the α and β subunits (M_r, 41857) were detected on Western blots. Restriction fragments of both clones were subcloned in pUC 8 and sequenced by the chain termination method. Thus the complete sequence of the genes of both subunits, hgdA (α) and hgdB (β) were obtained. The genes have the following order: A‐B, with an intergenic region of only 2 bp. The deduced amino acid sequences for the α and β subunits were confirmed by four peptides sequenced by protein chemical methods. Both chains are extremely rich in cysteine (13 in α, including a CNC and two CC clusters, and nine in (β) but no similarities to other known protein sequences were found.