Hyperactivity and interactions of a chimeric myristoryl-ACP thioesterase from the lux system of luminescent bacteria.
A chimeric myristoyl-ACP thioesterase with much higher catalytic efficiency than the parental enzymes has been generated by ligating the N-terminal half of the lux-specific thioesterase (LuxD) from Photobacterium phosphoreum with the C-terminal half of LuxD from Vibrio harveyi. The LuxD chimera had the same rate-limiting step and specificity, but cleaved esters and thioesters over eight times faster than the native enzymes. LuxD, along with acyl-protein synthetase (LuxE) and reductase (LuxC), comprise a multienzyme complex channeling activated fatty acids into the aldehyde substrate for the bacterial bioluminescence reaction. As P. phosphoreum LuxD and LuxE modulate each of their respective activities, the effects of mixing V. harveyi and the chimeric LuxD with P. phosphoreum LuxE were investigated. The chimeric LuxD stimulated acylation of LuxE to the same extent as V. harveyi LuxD, but to a lower level than that caused by P. phosphoreum LuxD. Conversely, P. phosphoreum LuxE stimulated the thioesterase activity of V. harveyi LuxD by 30% and the chimeric LuxD by 20% while the activity of P. phosphoreum LuxD was increased by over 140%. These results show that the stimulatory effects are unrelated to the level of thioesterase activity and indicate that the carboxyl terminal region of LuxD interacts with LuxE and causes a conformational change.
Li
J
J
Department of Biochemistry, Room 813, McIntyre Medical Sciences Building, McGill University, 3655 Promenade Sir William Osler, Que., H3G 1Y6, Montreal, Canada.
Szittner
R
R
Meighen
E A
EA
D000217:Acyltransferases / Q000737:chemistry; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry; D002678:Chimera; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008163:Luminescent Measurements; D008958:Models, Molecular; D010776:Photobacterium / Q000201:enzymology; D013869:Thiolester Hydrolases / Q000737:chemistry*; D014733:Vibrio / Q000201:enzymology
D001426:Bacterial Proteins; D000217:Acyltransferases; C034919:acyl protein synthetase; C065928:luxD protein, Bacteria; D013869:Thiolester Hydrolases; C018748:oleoyl-(acyl-carrier-protein) hydrolase