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Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases

  • Applied Microbial and Cell Physiology
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Abstract

Various hydroxyacyl coenzyme A (CoA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-14C]d-(−)-hydroxybutyric acid, [1-14C]d-lactic acid, [1-14C]l-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, d-(−)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5′-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-14C]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-14C]lactyl-CoA was used as a substrate at a rate that was only less than 10−4 of the rate than with [3-14C]d-(−)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-14C]d-lactyl-CoA as substrate at a relatively high rate.

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  1. Institut für Mikrobiologie der Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077, Göttingen, Germany

    Henry E. Valentin & Alexander Steinbüchel

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  1. Henry E. Valentin
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  2. Alexander Steinbüchel
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Correspondence to: A. Steinbüchel

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Valentin, H.E., Steinbüchel, A. Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases. Appl Microbiol Biotechnol 40, 699–709 (1994). https://doi.org/10.1007/BF00173332

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  • DOI: https://doi.org/10.1007/BF00173332

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