Construction and characterization of hybrid luciferases coded by lux genes from Xenorhabdus luminescens and Vibrio fischeri.
Molecular cloning techniques were employed to obtain hybrid luciferases with their alpha and beta subunits encoded by luxA and luxB genes, respectively, from Xenorhabdus luminescens strain HW or Vibrio fischeri. Although the two wild-type luminous bacteria are phylogenetically diverged, the hybrid luciferase Xf comprising an alpha from X. luminescens HW and a beta from V. fischeri and the hybrid luciferase VI comprising an alpha from V. fischeri and a beta from X. luminescens HW were both functional in bioluminescence. Their general kinetic properties were close to the wild-type enzymes from which the alpha subunit was derived. The X. luminescens HW enzyme is distinct in having a high optimal temperature for in vitro bioluminescence, a high thermal stability and a sensitivity to aldehyde substrate inhibition. Comparisons of the Xf and VI hybrid luciferases with the two wild-type enzymes indicated that these unusual properties of the X. luminescens HW luciferase originated primarily from the alpha subunit.
Department of Biochemical and Biophysical Sciences, University of Houston, TX 77204-5934.
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D001483:Base Sequence; D003001:Cloning, Molecular; D004755:Enterobacteriaceae / Q000201:enzymology* / Q000235:genetics*; D004795:Enzyme Stability; D005798:Genes, Bacterial; D007700:Kinetics; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D049449:Luminescence; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D055503:Protein Multimerization; D015183:Restriction Mapping; D013816:Thermodynamics; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics*
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D008156:Luciferases