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Saccharomyces Cerevisiae Oxidosqualene-Lanosterol Cyclase: a Chemistry-Biology Interdisciplinary Study of the Protein's Stru

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Saccharomyces Cerevisiae Oxidosqualene-Lanosterol Cyclase: a Chemistry-Biology Interdisciplinary Study of the Protein's Stru Saccharomyces cerevisiae Oxidosqualene- Lanosterol Cyclase: A Chemistry–Biology Interdisciplinary Study of the Protein’s THE CHEMICAL Structure–Function–Reaction Mechanism RECORD Relationships TUNG-KUNG WU, CHENG-HSIANG CHANG, YUAN-TING LIU, TSAI-TING WANG Department of Biological Science and Technology, National Chiao Tung University, 75, Po-Ai Street, Hsin-Chu 300, Taiwan, R.O.C. Received 15 June 2008; Accepted 1 August 2008 ABSTRACT: 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. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 302–325; 2008: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20157 Key words: oxidosqualene cyclase; biomimetic; site-saturated mutagenesis; molecular evolution; protein engineering; protein plasticity Introduction Oxidosqualene cyclases catalyze the enzymatic conversion of acids.1 In fungi and mammals, oxidosqualene is solely trans- an acyclic polyolefi n substrate, (3S)-2,3-oxidosqualene (OS), formed into a unique tetracyclic lanosterol, whereas a variety into a vast skeletal diversity of polycyclic sterols and triterpe- of polycyclic triterpene alcohols, including cycloartenol, lupeol, noids. In turn, the products serve as biosynthetic precursors or β-amyrin, are produced in higher plants and algae. In lower for sterols, related membranous components, steroid hor- plants and bacteria, squalene-hopene cyclase (SHC) converts mones, and secondary metabolites. The enzyme exhibits the precursor of oxidosqualene, squalene, into pentacyclic product specifi city in a species-dependent manner. Nearly 200 hopene or hopanol, which acts as corresponding sterol surro- distinct triterpenoid skeletons, mainly tetracycles and penta- gates of eukaryotic membranous triterpenoid lipid compo- cycles, have been isolated from various sources.1,2 The diverse nents (Scheme 1). product profi le is primarily controlled by the preorganized The intrinsic reaction complexity and product diversity of substrate conformation, regio- and stereocontrol of the ring oxidosqualene cyclase-catalyzed biotransformation have made skeleton, varied degrees of cyclization and rearrangement, and it one of the most exceptional biological reactions found in a fi nal deprotonation step at different positions through nature, and have fascinated both bioorganic chemists and bio- abstraction of a proton or addition of water molecules.3,4 Addi- chemists alike for over half a century, especially on the study tional dimensions of product diversity come from the enzy- matic oxidation or functional derivation with diverse auxiliary components such as sugars, amino acids, peptides, or fatty ᭤ Correspondence to: Tung-Kung Wu; e-mail: [email protected] The Chemical Record, Vol. 8, 302–325 (2008) 302 © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Protein’s Structure–Function–Reaction Mechanism of the enzyme-catalyzed cyclization/rearrangement reaction oxidosqualene-cycloartenol synthase (CAS)-catalyzed cycliza- mechanism.5,6 The proposed enzymatic mechanism of lanos- tion/rearrangement reactions, the substrate enters the enzyme terol synthesis involves a highly diverse and complex process active site in a prefolded chair–boat–chair (C-B-C) conforma- of substrate prefolding, acid-catalyzed epoxide protonation, tion, initiates and propagates the cyclization via an oxirane ring consecutive ring cyclization, hydride or methyl group rear- protonation and cleavage, to form a tetracyclic protosteryl rangement, and a fi nal deprotonation step (Scheme 2). In cation. This is followed by a series of 1,2-shifts of hydride and addition, the studies of oxidosqualene cyclase-catalyzed reac- methyl groups to the lanosteryl C-8/C-9 cation. Lanosterol is tions suggest that the cyclization and rearrangement sequences formed through abstracting a proton from lanosteryl C-8 or can break up to 16 bonds and form 16 new bonds (e.g., in the C-9 cationic intermediate, whereas cycloartenol is produced by conversion of OS to friedelin) as well as generate compounds deprotonation from the C-19 position and forming a cyclo- with up to 10 stereocenters in a single reaction (e.g., lupeol or propyl ring. On the other hand, the formation of triterpene taraxasterol).7 The remarkable product specifi city/diversity and alcohols, such as pentacyclic β-amyrin and lupeol, catalyzed by stereos-pecifi city is primarily controlled by the prefolded sub- β-amyrin synthase and lupeol synthase, respectively, proceeds strate conformation as well as the interactions between a series similarly except that the OS is prefolded into a chair–chair– of discrete, conformationally rigid, partially cyclized carboca- chair (C-C-C) substrate conformation, and proceeds with a tionic intermediates for deprotonation and the functional cationic cyclization to a dammarenyl cation, which is followed groups of the catalytic amino acid residues of the enzyme. by annulation of a fi fth ring and/or hydride migration. A For oxidosqualene-lanosterol cyclase (OSC or ERG7)- and generally accepted mechanism for the formation of lanosterol ᭤ Tung-Kung Wu was born in 1963 in Taiwan. He received his M.S. degree from the Chemistry Department of National Taiwan University under the supervision of Professor Tahsin J. Chow in 1988. In 1996, he received his Ph.D. degree in Biophysics from the Johns Hopkins University under the supervision of Professor Craig A. Townsend. From 1996 to 1998, he worked as a post- doctoral research fellow at Stanford University under the supervision of Professor John H. Griffi n. He was appointed assistant professor in the Department of Biological Science and Technology at the National Chiao Tung University in 1998, and was promoted to associate professor in 2004. His research interests include the interdisciplinary fi elds of organic chemistry, biochemistry, molecular biology, and genetics. His current research interests are mainly in the structure, function, mechanism, and biotechnological applications of oxidosqualene cyclases and molecular evolution of artifi cial enzymes for biosolar photovoltaic energy conversion. ᭿ ᭤ Cheng-Hsiang Chang was born in Taichung County, Taiwan, in 1977. He received his B.S. degree from the Department of Chemistry at National Chiao Tung University in Hisnchu City, Taiwan, in 2000. He then started his graduate studies on oxidosqualene cyclase under the supervi- sion of Professor Tung-Kung Wu in the Laboratory of Bioorganic Chemistry and Molecular Evolu- tion at the Department of Biological Science and Technology in the same school. During the fi rst year of his Ph.D. program, he studied the enzymology of oxidosqualene cyclase and, with his coworkers, established a purifi cation procedure for it from crude tissue. He received his Ph.D. degree in Biological Science and Technology. His research focuses on the functional characterization of oxidosqualene cyclases via various experimental strategies including mutagenesis approaches, inhibition studies, and SELEX technology. He is also interested in the fi elds of molecular biology and bioorganic chemistry and their relevance to the structural–functional relationships of cyclase enzymes. ᭿ © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. 303 THE CHEMICAL RECORD from oxidosqualene cyclase-catalyzed reactions is summarized catalytically important residues, to characterize the carboca- in Scheme 2. tionic intermediates involved in cyclization/rearrangement Early biomimetic studies on oxidosqualene cyclases, using cascade and to elucidate the structure–function–reaction radioisotope tracers and substrate analogs, have provided mechanism relationships. The goal of this report is to unravel important mechanistic insights regarding steric and functional the complexities of oxidosqualene cyclization/rearrangement tolerance of the substrate within the enzyme, as well as the cascade for rational design of cyclase inhibitors for hypocho- nature of the resulting products.3,4 However, detailed struc- lesterolemic, antifungal, and phytotoxic drugs, and to illumi- ture–function relationships for how enzyme functional resi- nate the process whereby protein engineering can generate new dues interact with substrates for preferentially promoting cyclases with novel or altered activity. certain cyclization, rearrangement, or deprotonation modes, await protein–ligand complex structure determination, or molecular-genetic selection coupled with product isolation and The Mechanistic and Stereochemical Insights for characterization. Oxidosqualene Cyclization In this paper, we historically review achievements by bio- mimetic studies and emphasize the current progress with Detailed mechanistic studies of oxidosqualene cyclization to regard to the Saccharomyces cerevisiae oxidosqualene-lanosterol form polycyclic products started with radioisotope tracer cyclase (SceERG7) enzyme. In this enzyme, site-directed and experiments performed by Woodward and Bloch
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