Conversion of glutaconate CoA-transferase from Acidaminococcus fermentans into an acyl-CoA hydrolase by site-directed mutagenesis

The heterooctameric (alpha beta)(4) glutaconate CoA-transferase (EC 2.8.3.12) from the anaerobic bacterium Acidamino-coccus fermentans catalyses the transfer of CoASH from actayl-CoA to the 1-carboxylate of glutaconate. During this reaction the glutamate residue 54 of the beta-subunit (beta E54) forms a CoA-ester. The single amino acid replacement beta E54D resulted in a drastic change of enzymatic function. The CoA-transferase activity decreased from 140 to less than 0.01 s(-1), whereas the acyl-CoA hydrolase activity increased from less than 0.01 to 16 s(-1). The new enzyme was able to catalyse the hydrolysis of glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA. Since the mutants beta E54A and beta 54N showed neither acyl-CoA hydrolase nor CoA-transferase activity, it was concluded that the aspartate carboxylate of the mutant beta E54D acted as a general base which facilitated the attack of water at the thiolester carbonyl. Surprisingly, K-m for glutaryl-CoA hydrolysis by the mutant (0.7 mu M) as compared to CoA-transfer by the wild-type (28 mu M) was 40 times lower. A 65 kDa protein, obtained by fusing the genes, gctA-gctB, coding for glutaconate CoA-transferase, retained 30% of the wild-type activity. Comparison of the amino acid sequences of 13 related enzymes demonstrated that Nature already has applied gene fusion in the case of pig heart CoA-transferase and has been using the E --> D mutation for catalysis by a yeast acetyl-CoA hydrolase. (C) 1997 Federation of European Biochemical Societies.