APPLICATION OF ENZYMATICALLY SYNTHESIZED SHORT-CHAIN-LENGTH HYDROXY FATTY-ACID COENZYME-A THIOESTERS FOR ASSAY OF POLYHYDROXYALKANOIC ACID SYNTHASES

Various hydroxyacyl coenzyme A (COA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-C-14]D-(-)-hyd roxybutyric acid, [1-C-14]D-lactic acid, [1-C-14]L-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostr idium propionicum. Preparative isolation of the thioesters on hydropho bic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalka noic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, D-(-)-3-hydroxybutyryl-CoA, which was detec ted spectroscopically at 412 nm by reduction of 5,5'-dithiobis(2-nitro benzoic acid) and provided a convenient assay of poly(3-hydroxybutyrat e) synthase. When [1-C-14]lactyl-CoA was used as substrate in a PHA sy nthase assay employing crude extracts obtained from various wild-type strains, [1-C-14]lactyl-CoA was used as a substrate at a rate that was only less than 10(-4) of the rate than with [3-C-14]D-(-)-3-hydroxybu tyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chr omatium vinosum and which used [1-C-14]d-lactyl-CoA as substrate at a relatively high rate.