Subunit interactions and the role of the luxA polypeptide in controlling thermal stability and catalytic properties in recombinant luciferase hybrids.
Bacterial luciferases with over 70% sequence identity from the terrestrial species, Xenorhabdus luminescens, and the marine species, Vibrio harveyi, exhibit large differences in thermal stability (Szittner and Meighen, 1990, J. Biol. Chem. 265, 16581-16587). The origin of these differences was investigated with genetically constructed hybrids containing one subunit from X. luminescens and the other from V. harveyi. While no activity was detected with the single (alpha and beta) subunits both in vitro and in vivo, the recombinant hybrid luciferases (alpha Xl beta Vh and alpha Vh beta Xh) were highly active and could be purified to homogeneity. The kinetic properties of the hybrid enzymes including aldehyde specificity, flavin binding and luminescence decay rates, were found to be nearly identical to those of the native luciferases (alpha Xl beta Xl or alpha Vh beta Vh) containing the same alpha subunit. In addition, the rate of thermal inactivation and temperature dependent quenching of the intrinsic fluorescence by flavin were found to be independent of the nature of the beta subunit, quite opposite to previous reports that the thermal stability is controlled by the beta subunit. Thus, the alpha subunit appears primarily responsible for controlling both the catalytic and structural properties of luciferase.
Li
Z
Z
Department of Biochemistry, McGill University Montreal, Canada.
Szittner
R
R
Meighen
E A
EA
D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli; D005486:Flavin Mononucleotide; D005798:Genes, Bacterial; D006358:Hot Temperature; D008156:Luciferases / Q000737:chemistry* / Q000302:isolation & purification; D008163:Luminescent Measurements; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D014733:Vibrio / Q000235:genetics
D001426:Bacterial Proteins; D010455:Peptides; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D008156:Luciferases