tailieunhanh - Báo cáo Y học: Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis

In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epivellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the a/b hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By sitedirected mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of. | Eur. J. Biochem. 269 2889-2896 2002 FEBS 2002 doi Potential active-site residues in polyneuridine aldehyde esterase a central enzyme of indole alkaloid biosynthesis by modelling and site-directed mutagenesis Emine Mattern-Dogru1 Xueyan Ma1 Joachim Hartmann2 Heinz Decker2 and Joachim Stockigt1 1Lehrstuhl fur Pharmazeutische Biologie Institut fur Pharmazie Johannes Gutenberg-Universitat Mainz Germany 2Lehrstuhlfur Molekulare Biophysik Johannes Gutenberg-Universităt Mainz Germany In the biosynthesis of the antiarrhythmic alkaloid ajmaline polyneuridine aldehyde esterase PNAE catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43 identity to a hydroxynitrile lyase from Hevea brasiliensis which is a member of the a b hydrolase superfamily. The catalytic triad which is typical for this family is conserved. By site-directed mutagenesis the members of the catalytic triad were identified. For further detection of the active residues a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model and were mutated in order to better understand the relationship of PNAE with the a b hydrolases and as well its mechanism of action. The results showed that PNAE is a novel member of the a b hydrolase enzyme superfamily. Keywords polyneuridine aldehyde esterase active-site residues site-directed mutagenesis modelling a b hydrolase enzyme superfamily. Polyneuridine aldehyde esterase PNAE EC is a central enzyme in a 10-step biosynthetic pathway expressed in the medicinal plant Rauvolfia serpentina Benth. ex Kurz. The pathway delivers the antiarrhythmic monoterpenoid indole alkaloid ajmaline 1 2 . From the enzymatic point of