Redesign of carnitine acetyltransferase specifity by protein engineering

Abstract

In eukaryotes, L-carnitine is involved in energy metabolism, where it facilitates b-oxidation of fatty acids. Carnitine acyltransferases catalyze the reversible conversion of acyl-CoA and carnitine to acylcarnitine and free CoA. There are three carnitine acyltransferase families, which differ in their acyl-chain length selectivity: carnitine palmitoyltransferases (CPTs) catalyze long-chain fatty acids, carnitine octanoyltransferase (COT) prefers medium-chain fatty acids, and carnitine acetyltransferase (CrAT) uses short-chain acyl-CoAs. <br/><br/>In this study, we attempted to identify the amino acid residues responsible for acyl-CoA specificity in the acyltransferase family through structure-based mutagenesis studies. We identified an amino acid (Met564 in rat CrAT) that is critical to fatty acyl chain-length specificity. A CrAT protein carrying the M564G mutation behaved as if its natural substrates were medium-chain acyl-CoAs, similar to COT. In the reverse case, mutation of the orthologous glycine (Gly553) to methionine in COT decreased activity towards its natural substrates, medium-chain acyl-CoAs, and increased activity towards short-chain acyl-CoAs. A second putative amino acid involved in acyl-CoA specificity was identified (Asp356 in rat CrAT), and the double CrAT mutant D356A/M564G behaved as a pseudo-CPT in terms of substrate specificity. Three-dimensional models revealed a deeper hydrophobic cavity for the binding of acyl groups in both CrAT mutants in the same position as the shallow cavity in the wt enzyme. Furthermore, we studied the effect of C75-CoA, a potent and competitive inhibitor of CPT I, on CrAT activity. C75-CoA occupies the same pocket in CPT I as palmitoyl-CoA, suggesting an inhibitory mechanism based on mutual exclusion. To determine whether this inhibitor would fit in the open hydrophobic pocket formed in CrAT mutants M564G and D356A/M564G, we carried out competitive inhibition assays. Our experiments showed that while C75-CoA is a potent inhibitor of CrAT mutants M564G and D356A/M564G, it has no effect on wt CrAT. <br/>Choline acetyltransferase (ChAT) catalyzes a similar reaction to CrAT, with the difference that in ChAT the acetyl group from acetyl-CoA is transferred to choline instead of carnitine. Cronin (1998) successfully redesigned ChAT to use carnitine instead of its natural substrate choline. In the present study, our aim was to achieve the opposite, that is, to redesign rat CrAT specificity from carnitine to choline. We prepared a mutant CrAT that incorporates four amino acid substitutions, and the resulting mutant shifted the catalytic discrimination between L-carnitine and choline in favour of the latter substrate. <br/>The food industry is interested in the production of esters for use as flavouring compounds; for example, esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced in an enzyme-catalyzed reaction between a higher alcohol and an acyl-CoA molecule. Since CrAT is responsible for the modulation of the acyl-CoA/CoA ratio, we hypothesized that overexpression of this enzyme could modify ester production in yeast. Therefore, we overexpressed CrAT in yeast and analysed its effect on ester production during alcoholic fermentation. Compared with control cells, overexpression of CrAT caused a significant reduction in the production of some esters, including the important flavour components ethyl acetate and 3-methyl-butyl acetate.<br/>In conclusion, the amino acid substitutions in rat CrAT and COT in this study reveal several residues that are involved in substrate recognition and provide insight into the molecular requirements for their correct positioning in order to achieve efficient catalysis. These results not only help us to understand the structure-function relationship within the acyltransferase family, but may also facilitate studies on obesity, non-insulin dependent diabetes, and patients with defective â-oxidation. Moreover, our results open the possibility of biotechnological applications of the enzymes of the carnitine acyltransferase family in the wine industry.