Enzymatic synthesis of 3-hydroxyacyl-CoAs of branched-chain amino acid catabolism.

Acyl-coenzyme A (CoA) thioesters occupy key positions in normal metabolism and are directly related to many inborn errors of metabolism. The degradation pathways of the branched-chain amino acids (BCAAs) are rich in acyl-CoA intermediates, many of which give rise to diagnostically important organic acids and acylcarnitines. Because several such acyl-CoAs are not routinely commercially available, they cannot be identified and quantified in biological samples. This leaves a gap in the characterization of BCAA-related inborn errors of metabolism. We attempted the enzymatic synthesis of BCAA-related 3-hydroxyacyl-CoAs, starting with the corresponding 2,3-enoyl free acids. First the 2,3-enoyl free acid is linked to CoA by purified recombinant glutaconate coenzyme A-transferase (GctAB), a bacterial CoA transferase active toward short chain acids. Then, hydration of the resulting 2,3-enoyl-acyl-CoA is catalyzed by recombinant human short-chain enoyl-CoA hydratase (ECHS1, gene ECHS1), producing a 3-hydroxyacyl-CoA. In this fashion, we synthesized 3-hydroxyisovaleryl-CoA, 3-hydroxyisobutyryl-CoA, 2-methyl-3-hydroxybutyryl-CoA and 3-hydroxypropionyl-CoA. All of these are detectable in normal mouse liver. We also found an unexpected peak with the same mass/charge ratio as 2-methyl-3-hydroxybutyryl-CoA. This proved to be 3-hydroxyvaleryl-CoA, an intermediate of odd chain fatty acid oxidation. All 3-hydroxyacyl-CoA intermediates of BCAA degradation are either commercially available or can be synthesized by the methods described.