Random and site-specific mutagenesis of bacterial luciferase : investigation of the substrate binding sites

Abstract

Luciferase from the Vibrio harveyi mutant AK-20 contains a lesion in the α subunit that results in decreased bioluminescence activity relative to the wild type. In addition, the AK-20 luciferase exhibits altered activities with different n-alkyl aldehydes. However, the thermal stability and substrate affinities of this mutant are similar to the wild type, suggesting an active center lesion. The luciferase structural genes (luxAB) from AK-20 were cloned into the vector pUC9 and expressed in E. coli. Only a single base change (C[to]T) was found in the luxA of AK-20. This base change translated into a Ser[to]Phe substitution at position α227 of the primary amino acid sequence. To determine if the AK-20 phenotype was due to the incorporation of the phenylalanine or the loss of the serine, site-specific mutagenesis of the luxA gene was carried out by substituting various amino acids at position 227. Several properties of the mutant luciferases were investigated and compared with those of the wild-type. These properties included bioluminescence activity with different n-alkyl aldehydes, substrate binding affinities and the thermal stabilities of the proteins and their intermediates II. The decay rates of the intermediates IIA and their dependence on aldehydes of different alkyl chainlengths were also investigated, since the decay of IIA was apparently the rate-limiting step in the bioluminescence reaction preceding light emission. The results showed that the luciferases containing large, relatively hydrophobic residues such as tyrosine, tryptophan and isoleucine possessed properties similar to AK-20 luciferase. Since the substitution of alanine, possessing a smaller, non-ionic side chain, and glycine both gave rise to luciferases with properties similar to the wild-type, it appeared that the substitution of phenylalanine at α277, rather than the loss of serine was the cause of the AK-20 phenotype. In addition, the fluorescence and chemical yields from the AK-20-catalyzed reaction were investigated to determine the reason for the lowered activity and quantum yield of the AK-20 luciferase. The results of these experiments demonstrated a dramatic decrease in the fluorescence intensity of the intermediate II as a possible source of the decreased bioluminescence activity of the AK-20 enzyme.