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.
Miller
David J
DJ
School of Chemistry, Main Building, Cardiff University, Park Place, Cardiff, UK CF10 3AT.
Gao
Jiali
J
Truhlar
Donald G
DG
Young
Neil J
NJ
Gonzalez
Veronica
V
Allemann
Rudolf K
RK
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