Cysteine-286 as the site of acylation of the Lux-specific fatty acyl-CoA reductase.
The channelling of fatty acids into the fatty aldehyde substrate for the bacterial bioluminescence reaction is catalyzed by a fatty acid reductase multienzyme complex, which channels fatty acids through the thioesterase (LuxD), synthetase (LuxE) and reductase (LuxC) components. Although all three components can be readily acylated in extracts of different luminescent bacteria, this complex has been successfully purified only from Photobacterium phosphoreum and the sites of acylation identified on LuxD and LuxE. To identify the acylation site on LuxC, the nucleotide sequence of P. phosphoreum luxC has been determined and the gene expressed in a mutant Escherichia coli strain. Even in crude extracts, the acylated reductase intermediate as well as acyl-CoA reductase activity could be readily detected, providing the basis for analysis of mutant reductases. Comparison of the amino-acid sequences of LuxC from P. phosphoreum, P. leiognathi and other luminescent bacteria, showed that only three cysteine residues (C171, C279, and C286) were conserved. As a cysteine residue on LuxC has been implicated in fatty acyl transfer, each of the conserved cysteine residues of the P. phosphoreum and P. leiognathi reductases was converted to a serine residue, and the properties of the mutant proteins examined. Only mutation of C286-blocked reductase activity and prevented formation of the acylated reductase intermediate, showing that C286 is the site of acylation on LuxC.
Lee
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CY
Department of Biochemistry, McGill University, Montreal, Que., Canada.
Meighen
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EA
D000215:Acylation; D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D010088:Oxidoreductases; D010776:Photobacterium / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D010088:Oxidoreductases; D000445:Aldehyde Oxidoreductases; C021351:hexadecanal dehydrogenase (acylating); C055198:LuxC protein, Bacteria; D003545:Cysteine