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 terrest rial 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 d ifferences was investigated with genetically constructed hybrids conta ining one subunit from X. luminescens and the other from V harveyi. Wh ile 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 pur ified to homogeneity. The kinetic properties of the hybrid enzymes inc luding aldehyde specificity, flavin binding and luminescence decay rat es, were found to be nearly identical to those of the native luciferas es (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 foun d to be independent of the nature of the beta subunit, quite opposite to previous reports that the thermal stability is controlled by the be ta subunit. Thus, the alpha subunit appears primarily responsible for controlling both the catalytic and structural properties of luciferase .