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 ha lf of the lux-specific thioesterase (LuxD) from Photobacterium phosphoreum with the C-terminal half of LuxD from Vibrio harveyi The LuxD chimera had t he same rate-limiting step and specificity, but cleaved esters and thioeste rs over eight times faster than the native enzymes. LuxD, along with acyl-p rotein synthetase (LuxE) and reductase (LuxC), comprise a multienzyme compl ex channeling activated fatty acids into the aldehyde substrate for the bac terial bioluminescence reaction. As P. phosphoreum LuxD and LuxE modulate e ach of their respective activities, the effects of mixing V. halveyi and th e chimeric LuxD with P. phosphoreum LuxE were investigated. The chimeric Lu xD 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. ph osphoreum LuxE stimulated the thioesterase activity of V. harveyi LuxD by 3 0% and the chimeric LuxD by 20% while the activity of P. phosphoreum LuxD w as increased by over 140%. These results show that the stimulatory effects are unrelated to the level of thioesterase activity and indicate that the c arboxyl terminal region of LuxD interacts with LuxE and causes a conformati onal change. (C) 2000 Elsevier Science B.V. All rights reserved.