Effects of 3' end deletions from the Vibrio harveyi luxB gene on luciferase subunit folding and enzyme assembly: generation of temperature-sensitive polypeptide folding mutants

Abstract

Ten recombinant plasmids have been constructed by deletion of specific regions from the plasmid pTB7 that carries the luxA and luxB genes, encoding the .alpha. and .beta. subunits of luciferase from Vibrio harveyi, such that luciferases with normal .alpha. subunits and variant .beta. subunits were produced in Escherichia coli cells carrying the recombinant plasmids. The original plasmid, which conferred bioluminescence (upon addition of exogenous aldehyde substrate) on E. coli carrying it, was constructed by insertion of a 4.0-kb HindIII fragment of V. harveyi DNA into the HindIII site of plasmid pBR322 [Baldwin, T. O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., and Ziegler, M. M. (1984) Biochemistry 23, 3663-3667]. Deletion mutants in the 3'' region of luxB were divided into three groups: (A) those with deletions in the 3'' untranslated region that left the coding sequences intact, (B) those that left the 3'' untranslated sequences intact but deleted short stretches of the 3'' coding region of the .beta. subunit, and (C) those for which the 3'' deletions extended from the untranslated region into the coding sequences. Analysis of the expression of luciferase from these variant plasmids has demonstrated two points concerning the synthesis of luciferase subunits and the assembly of those subunits into active luciferase in E. coli. First, deletion of DNA sequences 3'' to the translational open reading frame of the .beta. subunit that contain a potential stem and loop structure resulted in dramatic reduction in the level of accumulation of active luciferase in cells carrying the variant plasmids, even though the luxAB coding regions remained intact. Second, the C-terminal ca. 10-15 residues of the .beta. subunit appeared to have little to do with the structure or stability of the active heterodimeric form of the luciferase, but deletion of amino acid residues from this region resulted in greatly reduced levels of accumulation of active heterodimeric luciferase, especially at higher temperatures. The active luciferase that did form from the truncated .beta. subunit (.beta.t) constuctions had essentially normal activity and stability but impaired ability to refold from urea. As with the apparent temperature sensitivity of accumulation of active luciferase in vivo, these variants showed temperature sensitivity in refolding from urea in vitro. We conclude that the carboxyl-terminal region of the .beta. subunit has little to do with the bioluminescence reaction or stability of the dimeric structure per se, but it does appear to play a critical function in proper folding and/or assembly into the active dimeric structure.