Conversion of glutaconate CoA-transferase from Acidaminococcus fermentans into an acyl-CoA hydrolase by site-directed mutagenesis.
The heterooctameric (alphabeta)4 glutaconate CoA-transferase (EC 2.8.3.12) from the anaerobic bacterium Acidaminococcus fermentans catalyses the transfer of CoASH from acetyl-CoA to the 1-carboxylate of glutaconate. During this reaction the glutamate residue 54 of the beta-subunit (betaE54) forms a CoA-ester. The single amino acid replacement betaE54D 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 betaE54A and betaE54N showed neither acyl-CoA hydrolase nor CoA-transferase activity, it was concluded that the aspartate carboxylate of the mutant betaE54D acted as a general base which facilitated the attack of water at the thiolester carbonyl. Surprisingly, Km for glutaryl-CoA hydrolysis by the mutant (0.7 microM) as compared to CoA-transfer by the wild-type (28 microM) 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.
Mack
M
M
Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität Marburg, Germany.
Buckel
W
W
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015256:Coenzyme A-Transferases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D018698:Glutamic Acid / Q000235:genetics; D016964:Gram-Negative Anaerobic Cocci / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010170:Palmitoyl-CoA Hydrolase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D018698:Glutamic Acid; D015256:Coenzyme A-Transferases; C032430:glutaconate CoA-transferase; D010170:Palmitoyl-CoA Hydrolase