Lux C, D and E genes of the Vibrio fischeri luminescence operon code for the reductase, transferase, and synthetase enzymes involved in aldehyde biosynthesis.
The lux C, D, and E genes of the Vibrio fischeri luminescence operon code for three polypeptides of 54, 33, and 42 kDa, respectively, which are required for synthesis of the aldehyde substrate for the luminescent reaction. These polypeptides have been identified in V. fischeri and V. harveyi as well as in recombinant E. coli harboring the cloned genes by specific acylation with [3H]fatty acid, showing that they are components of a fatty acid reductase system with reductase, synthetase and transferase activities. By using glycerol in the assay and/or extraction buffer and decreasing the reducing agent, the levels of the acylation of the 54 and 42 kDa polypeptides have been greatly increased. As a consequence, it was possible to demonstrate that the 54 kDa polypeptide coded by the lux C gene has reductase activity. In a subclone missing the lux E gene, the 42 kDa polypeptide was missing and the 54 kDa polypeptide could not be acylated in vitro with tetradecanoic acid (+ATP) and only to a low level in vivo indicating that the synthetase enzyme, responsible for fatty acid activation, is coded by the lux E gene. In vitro acylation with tetradecanoyl CoA of the 33 kDa polypeptide coupled with the specific cleavage of acyl-ACP only in E. coli extracts transformed with DNA containing the lux D gene, demonstrated that the lux D gene coded for the transferase enzyme.
M
M
Boylan
C
C
Miyamoto
L
L
Wall
A
A
Graham
E
E
Meighen
D000447:Aldehydes; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D008025:Ligases / Q000235:genetics*; D049449:Luminescence; D009876:Operon; D010088:Oxidoreductases / Q000235:genetics*; D014166:Transferases / Q000235:genetics*; D014733:Vibrio / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
D000447:Aldehydes; D010088:Oxidoreductases; D014166:Transferases; D008025:Ligases