title: Probing the Vibrio harveyi luciferase beta subunit functionality and the intersubunit domain by site-directed mutagenesis.
abstract: While the critical role of the bacterial luciferase alpha subunit in catalysis has been amply documented, the beta subunit was only known to be involved in thermal stability and substrate binding. Two conserved histidyl residues at position 81 and 82 of the beta subunit of Vibrio harveyi luciferase were each mutated to an alanine, aspartate, or lysine to probe further the beta functionality. These mutations resulted in higher Km values for reduced riboflavin 5'-phosphate, less efficient oxidations of the aldehyde substrate, and decreased light-emitting activities. beta His82 appears to be significantly more critical than beta His81. For the beta His82-mutated luciferases, the maximal light intensities and total light outputs were reduced to 19-4% of that for the wild-type enzyme, and the values of Vmax/Km,flavin were decreased by 2-3 orders of magnitude. The reduced light emission activities for these mutated luciferases can be correlated to lower yields of the flavin 4a-hydroperoxide intermediate, reduced productions of the excited flavin emitter, and/or enhanced quenching of the emitter. The beta subunit and the conserved beta His82 in particular have thus been shown to be critical not only to flavin binding but also to catalytic characteristics of luciferase. The dimeric structure of luciferase is essential to its high catalytic efficiency. To characterize the intersubunit domain, three sets of single/double mutants were constructed, and the additivities of mutational effects were tested to screen for residues that could interact across the subunit interface.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5934.
authors.forename: X
authors.initials: X
authors.lastname: Xin
authors.forename: L
authors.initials: L
authors.lastname: Xi
authors.forename: S C
authors.initials: SC
authors.lastname: Tu
mesh_terms: D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014733:Vibrio / Q000201:enzymology*
chemical_list: D006639:Histidine; D008156:Luciferases