A stereochemical study on the metabolism of isobutyrate in Pseudomonas putida

Ammonium[2- 3 H,1- 14 C]isobutyrate was converted by Pseudomonas putida ATCC 21244 into S(+)-β-hydroxyisobutyric acid (β-HIBA) with loss of the α-tritium atom. The recovered isobutyrate had the same 3 H 14 C as the starting material. Ammonium (2 S )-[3- 13 C]isobutyrate was synthesized and converted by P. putida into β-HIBA. The 13 C-nmr of the corresponding methyl ester benzoate showed 13 C enrichment in the hydroxymethyl carbon atom. The results therefore indicate that isobutyrate metabolism in this organism proceeds via an unsaturated intermediate (probably methacrylyl-CoA) formed by dehydrogenation of the 2- pro-S -methyl group of the precursor (isobutyryl-CoA). Hydration of the intermediate proceeds with addition of a proton at C-2 from the same side as the hydrogen removed in the dehydrogenation.