Site-saturated mutagenesis experiments were carried out on the His234 residue of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7) to characterize its functional role in ERG7 activity and to determine its effect on the oxidosqualene cyclization/rearrangement reaction. Two novel intermediates, (13alphaH)-isomalabarica-14(26),17E,21-trien-3beta-ol and protosta-20,24-dien-3beta-ol, isolated from ERG7(H234X) mutants, provided direct mechanistic evidence for formation of the chair-boat 6-6-5 tricyclic Markovnikov cation and protosteryl cation that were assigned provisionally to the ERG7-catalyzed biosynthetic pathway. In addition, we obtained mutants that showed a complete change in product specificity from lanosterol formation to either protosta-12,24-dien-3beta-ol or parkeol production. Finally, the repeated observation of multiple abortive and/or alternative cyclization/arrangement products from various ERG7(H234X) mutants demonstrated the catalytic plasticity of the enzyme. Specifically, subtle changes in the active site affect both the stability of the cation-pi interaction and generate product diversity.

Oxidosqualene cyclases catalyze the biotransformation of acyclic (3S)-2,3-oxidosqualene (OS) to a variety of polycyclic sterols and triterpenoids, generating over 100 distinct triterpenoid skeletons with the formula C 30 H 50 O.[1-3, 4 and references therein] Product specificity is species-dependent and precisely controlled by the prefolded substrate conformation as well as by interactions between the carbocationic intermediate for deprotonation and the functional groups of catalytic amino acid residues of the enzyme. The transformation mechanisms of this single class of enzymes can vary widely. For example, the triterpenes lanosterol, cycloartenol, and parkeol are formed from a preorganized chair-boat-chair substrate conformation of OS, and cationic cyclization to the protosteryl cation is followed by skeletal rearrangements until the final deprotonation step. Formation of the pentacyclic b-amyrin and lupeol proceed similarly except that OS is in the chair-chair-chair conformation (this results in stereochemical differences in the products relative to the chair-boat-chair substrate conformation), and the cationic cyclization to the dammarenyl cation is followed by annulation of a fifth ring.Various strategies have been used to probe the complex cyclization/rearrangement reaction mechanism, both for the purpose of understanding these complex enzymes and also to engineer cyclases to generate new product profiles. For example, site-directed mutagenesis was used to identify the residues responsible for the product specificity of b-amyrin synthase (PNY) and lupeol synthase (OEW). Two residues of PNY from Panax ginseng, Trp259 and Tyr261, were found to play important roles in the reaction mechanism to direct b-amyrin and/or lupeol formation.[5] We and others independently identified several critical residues from oxidosqualene-lanosterol cyclase (ERG7) from Saccharomyces cerevisiae and oxidosqualenecycloartenol synthase (CAS) from Arabidopsis thaliana, and demonstrated their roles in facilitating tetracyclic formation and/or stabilizing the lanosteryl cation for deprotonation, as [a] Prof.

[reaction: see text] We describe the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase Phe445 site-saturated mutants that generate truncated tricyclic and altered deprotonation product profiles. Among these mutants, only polar side-chain group substitutions genetically complemented yeast viability and produced spatially related product diversity, supporting the Johnson model that cation-pi interactions between a carbocationic intermediate and an enzyme can be replaced by an electrostatic or polar side chain to stabilize the cationic intermediate, but with product differentiation.

Glycosyltransferase 1 from Bacillus cereus (BcGT1) catalyzes the transfer of a glucosyl moiety from uridine diphosphate glucose (UDP-glucose) to various acceptors; it was expressed and characterized. The specificity of acceptors was found to be broad: more than 20 compounds classified into O-, S-, and N-linkage glucosides can be prepared with BcGT1 catalysis. Based on this work, we conclude that the corresponding acceptors of these compounds must possess the following features: (1) the acceptors must contain at least one aromatic or fused-aromatic or heteroaromatic ring; (2) the reactive hydroxyl or sulfhydryl or amino group can attach either on the aromatic ring or on its aliphatic side chain; and (3) the acceptors can be a primary, secondary, or even a tertiary amine. Four representative acceptors-fluorescein methyl ester, 17-β-estradiol, 7-mercapto-4-methylcoumarin, and 6-benzylaminopurine-were chosen as a candidate acceptor for O-, S-, and N-glucosidation, respectively. These enzymatic products were purified and the structures were confirmed with mass and NMR spectra. As all isolated glucosides are β-anomers, BcGT1 is confirmed to be an inverting enzyme. This study not only demonstrates the substrate promiscuity of BcGT1 but also showed the great application prospect of this enzyme in bioconversion of valuable bioactive molecules.

The oxidosqualene cyclases (EC 5.4.99-) constitute a family of enzymes that catalyze diverse cyclization/rearrangement reactions of (3S)-2,3-oxidosqualene into a distinct array of sterols and triterpenes. The relationship between the cyclization mechanism and the enzymatic structure is extremely complex and compelling. This review covers the historical achievements of biomimetic studies and current progress in structural biology, molecular genetics, and bioinformatics studies to elucidate the mechanistic and structure-function relationships of the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase-catalyzed cyclization/rearrangement reaction.