The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes.
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 multienzyme 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.
Kater
M M
MM
Department of Genetics, BioCentrum Amsterdam, Vrije Universiteit, Netherlands.
Koningstein
G M
GM
Nijkamp
H J
HJ
Stuitje
A R
AR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001896:Boron Compounds / Q000494:pharmacology; D001937:Brassica / Q000235:genetics; D004274:DNA, Recombinant; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D064429:Fatty Acid Synthases / Q000235:genetics*; D005227:Fatty Acids / Q000032:analysis; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009829:Oleic Acids / Q000096:biosynthesis; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid
D001896:Boron Compounds; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D005227:Fatty Acids; D005229:Fatty Acids, Monounsaturated; D009829:Oleic Acids; D010743:Phospholipids; C008757:palmitoleic acid; C065593:cis-vaccenic acid; C032255:1,2-dihydro-1-hydroxy-6-methyl-2-(propanesulfonyl)-thieno(3,2D)(1,2,3)-diazaborine; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); C493924:fabI protein, E coli; D064429:Fatty Acid Synthases; D054889:Fatty Acid Synthase, Type II