TY - JOUR
T1 - Stereochemistry of eudesmane cation formation during catalysis by aristolochene synthase from Penicillium roqueforti
AU - Miller, David J.
AU - Gao, Jiali
AU - Truhlar, Donald G.
AU - Young, Neil J.
AU - Gonzalez, Veronica
AU - Allemann, Rudolf K.
PY - 2008
Y1 - 2008
N2 - The aristolochene synthase catalysed cyclisation of farnesyl diphosphate (1) has been postulated to proceed through (S)-germacrene A (3). However, the active site acid that reprotonates this neutral intermediate has so far proved difficult to identify and, based on high level ab initio molecular orbital and density functional theory calculations, a proton transfer mechanism has recently been proposed, in which proton transfer from C12 of germacryl cation to the C6,C7-double bond of germacryl cation (2) proceeds either directly or via a tightly bound water molecule. In this work, the stereochemistry of the elimination and protonation reactions was investigated by the analysis of the reaction products from incubation of 1 and of [12,12,12,13,13,13- 2H6]-farnesyl diphosphate (15) with aristolochene synthase from Penicillium roqueforti (PR-AS) in H2O and D2O. The results reveal proton loss from C12 during the reaction and incorporation of another proton from the solvent. Incubation of 1 with PR-AS in D2O led to the production of (6R)-[6-2H] aristolochene, indicating that protonation occurs from the face of the 10-membered germacrene ring opposite the isopropylidene group. Hence these results firmly exclude proton transfer from C12 to C6 of germacryl cation. We propose here Lys 206 as the general acid/base during PR-AS catalysis. This residue is part of a conserved network of hydrogen bonds, along which protons could be delivered from the solvent to the active site.
AB - The aristolochene synthase catalysed cyclisation of farnesyl diphosphate (1) has been postulated to proceed through (S)-germacrene A (3). However, the active site acid that reprotonates this neutral intermediate has so far proved difficult to identify and, based on high level ab initio molecular orbital and density functional theory calculations, a proton transfer mechanism has recently been proposed, in which proton transfer from C12 of germacryl cation to the C6,C7-double bond of germacryl cation (2) proceeds either directly or via a tightly bound water molecule. In this work, the stereochemistry of the elimination and protonation reactions was investigated by the analysis of the reaction products from incubation of 1 and of [12,12,12,13,13,13- 2H6]-farnesyl diphosphate (15) with aristolochene synthase from Penicillium roqueforti (PR-AS) in H2O and D2O. The results reveal proton loss from C12 during the reaction and incorporation of another proton from the solvent. Incubation of 1 with PR-AS in D2O led to the production of (6R)-[6-2H] aristolochene, indicating that protonation occurs from the face of the 10-membered germacrene ring opposite the isopropylidene group. Hence these results firmly exclude proton transfer from C12 to C6 of germacryl cation. We propose here Lys 206 as the general acid/base during PR-AS catalysis. This residue is part of a conserved network of hydrogen bonds, along which protons could be delivered from the solvent to the active site.
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U2 - 10.1039/b804198a
DO - 10.1039/b804198a
M3 - Article
C2 - 18563268
AN - SCOPUS:45749137172
SN - 1477-0520
VL - 6
SP - 2346
EP - 2354
JO - Organic and Biomolecular Chemistry
JF - Organic and Biomolecular Chemistry
IS - 13
ER -