Functional consequences of site-directed mutation of conserved histidyl residues of the bacterial luciferase alpha subunit.
The available sequences for the different bacterial luciferases reveal five conserved histidyl residues at positions 44, 45, 82, 224, and 285 of the alpha subunit. Ten variants of Vibrio harveyi luciferase were obtained by selective site-directed mutations of these five histidines. The essentiality of alpha His44 and alpha His45 was indicated by 4-7 orders of magnitude of bioluminescence activity reductions resulting from the substitution of either histidine by alanine (alpha H44A or alpha H45A), aspartate (alpha H44D or alpha H45D), or lysine (alpha H45K). Moreover, alpha H44A and alpha H45A were distinct from the native luciferase in thermal stabilities. Mutations at the other three positions also resulted in activity reductions ranging from a fewfold to 3 orders of magnitude. Despite these widely different bioluminescence light outputs, mutated luciferases exhibited, in nonturnover in vitro assays, light emission decay rates mostly similar to that of the native luciferase using octanal, decanal, or dodecanal as a substrate. This is attributed to a similarity in the catalytic rate constants of the light-emitting pathway for the native and mutated luciferases, but various mutated luciferases suffer in different degrees from competing dark reaction(s). In accord with this interpretation, the bioluminescence activities of mutated luciferases showed a general parallel with the relative stabilities of their 4a-hydroperoxyflavin intermediate species. Furthermore, the drastically reduced bioluminescence activities for luciferases with the alpha His44 or alpha His45 substituted by aspartate, alanine, or lysine were accompanied by little or no activities for consuming the aldehyde substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5934.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004795:Enzyme Stability; D006639:Histidine; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D003062:Codon; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006639:Histidine; D008156:Luciferases