The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase
1994
Kater, M.M. | Koningstein, G.M. | Nijkamp, H.J.J. | Stuitje, A.R.
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 the envM 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 an envM mutant of Escherichia 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 multi-enzyme complex. Finally, lipid analysis of recombinant E. 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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