Control of luminescence decay and flavin binding by the LuxA carboxyl-terminal regions in chimeric bacterial luciferases.

Abstract

Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as P. (Vibrio) fischeri, P. phosphoreum, and P. leiognathi, have rapid decay rates. By generation of an X. luminescens-based chimeric luciferase with a 67 amino acid substitution from P. phosphoreum LuxA in the central region of the LuxA subunit, the "slow" X. luminescens luciferase was converted into a chimeric luciferase, LuxA(1)B, with a significantly more rapid decay rate. Two other chimeras with P. phosphoreum sequences substituted closer to the carboxyl terminal of LuxA, LuxA(2)B and LuxA(3)B, retained the characteristic slow decay rates of X. luminescens luciferase but had weaker interactions with both reduced and oxidized flavins, implicating the carboxyl-terminal regions in flavin binding. The dependence of the luminescence decay on concentration and type of fatty aldehyde indicated that the decay rate of "fast" luciferases arose due to a high dissociation constant (K(a)) for aldehyde (A) coupled with the rapid decay of the resultant aldehyde-free complex via a dark pathway. The decay rate of luminescence (k(T)) was related to the decanal concentration by the equation: k(T) = (k(L)A + k(D)K(a))/(K(a) + A), showing that the rate constant for luminescence decay is equal to the decay rate via the dark- (k(D)) and light-emitting (k(L)) pathways at low and high aldehyde concentrations, respectively. These results strongly implicate the central region in LuxA(1)B as critical in differentiating between "slow" and "fast" luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase-flavin-oxygen intermediate.