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 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 alpha/beta 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 alpha/beta hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the alpha/beta hydrolase enzyme superfamily.
Mattern-Dogru
Emine
E
Lehrstuhl für Pharmazeutische Biologie, Institut für Pharmazie, Johannes Gutenberg-Universität Mainz, Germany.
Ma
Xueyan
X
Hartmann
Joachim
J
Decker
Heinz
H
Stöckigt
Joachim
J
D000097:Acetonitriles / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D028482:Hevea / Q000201:enzymology; D026121:Indole Alkaloids / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011926:Rauwolfia / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000097:Acetonitriles; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; C045516:mandelonitrile; D002265:Carboxylic Ester Hydrolases; C463439:polyneuridine aldehyde esterase; D000446:Aldehyde-Lyases; C079359:hydroxymandelonitrile lyase