The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes |
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Authors: | Martin M. Kater Gregory M. Koningstein H. John J. Nijkamp Antoine R. Stuitje |
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Affiliation: | (1) Department of Genetics, Institute for Molecular Biological Sciences, BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, Netherlands;(2) Present address: Department of Developmental Biology, DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), P.O. Box 16, 6700 AA Wageningen, The Netherlands |
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Abstract: | Fatty acid synthesis in bacteria and plants is catalysed by a multi-enzyme fatty acid synthetase complex (FAS II) which consists of separate monofunctional polypeptides. Here we present a comparative molecular genetic and biochemical study of the enoyl-ACP reductase FAS components of plant and bacterial origin. The putative bacterial enoyl-ACP reductase gene (envM) was identified on the basis of amino acid sequence similarities with the recently cloned plant enoyl-ACP reductase. Subsequently, it was unambiguously demonstrated by overexpression studies that theenvM gene encodes the bacterial enoyl-ACP reductase. An anti-bacterial agent called diazaborine was shown to be a specific inhibitor of the bacterial enoyl-ACP reductase, whereas the plant enzyme was insensitive to this synthetic antibiotic. The close functional relationship between the plant and bacterial enoyl-ACP reductases was inferred from genetic complementation of anenvM mutant ofEscherichia coli. Ultimately,envM gene-replacement studies, facilitated by the use of diazaborine, demonstrated for the first time that a single component of the plant FAS system can functionally replace its counterpart within the bacterial multienzyme complex. Finally, lipid analysis of recombinantE. coli strains with the hybrid FAS system unexpectedly revealed that enoyl-ACP reductase catalyses a rate-limiting step in the elongation of unsaturated fatty acids. |
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Keywords: | Escherichia coli envM gene enoyl-ACP reductase fatty acid synthetase gene replacement diazaborine |
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