Stereochemistry of eudesmane cation formation during catalysis by aristolochene synthase from Penicillium roqueforti.
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-(2)H(6)]-farnesyl diphosphate (15) with aristolochene synthase from Penicillium roqueforti (PR-AS) in H(2)O and D(2)O. The results reveal proton loss from C12 during the reaction and incorporation of another proton from the solvent. Incubation of with PR-AS in D(2)O led to the production of (6R)-[6-(2)H] 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.
School of Chemistry, Main Building, Cardiff University, Park Place, Cardiff, UK CF10 3AT.
David J
DJ
Miller
Jiali
J
Gao
Donald G
DG
Truhlar
Neil J
NJ
Young
Veronica
V
Gonzalez
Rudolf K
RK
Allemann
D002384:Catalysis; D003500:Cyclization; D005740:Gases / Q000737:chemistry; D007535:Isomerases / Q000378:metabolism*; D010407:Penicillium / Q000201:enzymology*; D011522:Protons; D045787:Sesquiterpenes, Eudesmane / Q000737:chemistry* / Q000378:metabolism*; D013237:Stereoisomerism; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
D005740:Gases; D011522:Protons; D045787:Sesquiterpenes, Eudesmane; D014867:Water; C015872:eudesmane; D007535:Isomerases; C060050:aristolochene synthase