Changes in the Kinetics and Emission Spectrum on Mutation of the Chromophore-Binding Platform in Vibrio harveyi Luciferase
详细信息 查看全文
- 作者：Leo Yen-Cheng Lin ; Rose Szittner ; Romy Friedman ; and Edward A. Meighen
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：March 23, 2004
- 年：2004
- 卷：43
- 期：11
- 页码：3183 - 3194
- 全文大小：375K
- 年卷期：v.43,no.11(March 23, 2004)
- ISSN：1520-4995
文摘
The recently proposed model for the bacteria luciferase-flavin mononucleotide complexidentifies a number of critical intermolecular interactions that define a binding platform for the isoalloxazinering of flavin [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A.(2001) Protein Sci. 10, 1563-1571]. A key interaction involving van der Waals contact between theisopropyl side chain of 
Val173 and the 7,8-dimethyl benzene plane of the isoalloxazine chromophorerepresents an important target to test the validity of the proposed model. Here, structure-function analysisof luciferase variants carrying single point mutations at position 173 have verified the functional layoutof the active site architecture and implicated this site directly in flavin binding. Moreover, a decrease inthe stability of the enzyme-bound C4a-hydroperoxyflavin intermediate in the mutants could account forchanges in saturation with the fatty aldehyde substrate. A predicted red-shift on mutation of position173 to increase its polarity confirmed that Val173 was an integral component of the chromophore-binding microenvironment. Introduction of mutations in residues that contact the pyrimidine plane of theisoalloxazine chromophore (A75G/C106V) into the V173A, V173C, V173T, and V173S mutantsled to the retention of high levels of enzyme activity (10-40% of wild type) and further red-shifted theemission spectra in the triple mutants. The additivity of the mutation-induced red-shifts in the emissionwavelength spectrum provides the basis toward engineering luciferase variants that emit different lightcolors with the proposed flavin-luciferase model complex as a design reference.
Val173 and the 7,8-dimethyl benzene plane of the isoalloxazine chromophorerepresents an important target to test the validity of the proposed model. Here, structure-function analysisof luciferase variants carrying single point mutations at position 173 have verified the functional layoutof the active site architecture and implicated this site directly in flavin binding. Moreover, a decrease inthe stability of the enzyme-bound C4a-hydroperoxyflavin intermediate in the mutants could account forchanges in saturation with the fatty aldehyde substrate. A predicted red-shift on mutation of position173 to increase its polarity confirmed that Val173 was an integral component of the chromophore-binding microenvironment. Introduction of mutations in residues that contact the pyrimidine plane of theisoalloxazine chromophore (A75G/C106V) into the V173A, V173C, V173T, and V173S mutantsled to the retention of high levels of enzyme activity (10-40% of wild type) and further red-shifted theemission spectra in the triple mutants. The additivity of the mutation-induced red-shifts in the emissionwavelength spectrum provides the basis toward engineering luciferase variants that emit different lightcolors with the proposed flavin-luciferase model complex as a design reference.