Control of Luminescence Decay and Flavin Binding by the LuxA Carboxyl-Terminal Regions in Chimeric Bacterial Luciferases

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• 作者：Nelly Valkova ; Rose Szittner ; and Edward A. Meighen
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：October 19, 1999
• 年：1999
• 卷：38
• 期：42
• 页码：13820 - 13828
• 全文大小：325K
• 年卷期：v.38,no.42(October 19, 1999)
• ISSN：1520-4995

文摘

Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases basedon their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi andXenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacteriumgenus, such as P. (Vibrio) fischeri, P. phosphoreum, and P. leiognathi, have rapid decay rates. By generationof an X. luminescens-based chimeric luciferase with a 67 amino acid substitution from P. phosphoreumLuxA in the central region of the LuxA subunit, the "slow" X. luminescens luciferase was converted intoa chimeric luciferase, LuxA₁B, with a significantly more rapid decay rate. Two other chimeras with P.phosphoreum sequences substituted closer to the carboxyl terminal of LuxA, LuxA₂B and LuxA₃B, retainedthe characteristic slow decay rates of X. luminescens luciferase but had weaker interactions with bothreduced and oxidized flavins, implicating the carboxyl-terminal regions in flavin binding. The dependenceof 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 rapiddecay 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_LA + k_DK_a)/(K_a + A), showing that therate 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 centralregion in LuxA₁B as critical in differentiating between "slow" and "fast" luciferases and show that thisdistinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase-flavin-oxygen intermediate.