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1202 Biochemical Society Transactions (2005) Volume 33, part 5 Access of the substrate to the active site of squalene and oxidosqualene cyclases: comparative inhibition, site-directed mutagenesis and homology-modelling studies S. Oliaro-Bosso*, T. Schulz-Gasch†, S. Taramino*, M. Scaldaferri*, F. Viola* and G. Balliano*1 *Dipartimento di Scienza e Tecnologia del Farmaco, Facoltà di Farmacia, Università degli Studi di Torino, Via Pietro Giuria 9, I-10125 Turin, Italy, and †F. Hoffmann-La Roche Ltd., Molecular Design, PRBD-CS 92/2.10D, CH-4010 Basel, Switzerland Abstract Substrate access to the active-site cavity of squalene-hopene cyclase from Alicyclobacillus acidocaldarious and lanosterol synthase [OSC (oxidosqualene cyclase)] from Saccharomyces cerevisiae was studied by an inhibition, mutagenesis and homology-modelling approach. Crystal structure and homology modelling indicate that both enzymes possess a narrow constriction that separates an entrance lipophilic channel from the active-site cavity. The role of the constriction as a mobile gate that permits substrate passage was investigated by experiments in which critically located Cys residues, either present in native protein or inserted by site-directed mutagenesis, were labelled with specifically designed thiol-reacting molecules. Some amino acid residues of the yeast enzyme, selected on the basis of sequence alignment and a homology model, were individually replaced by residues bearing side chains of different lengths, charges or hydrophobicities. In some of these mutants, substitution severely reduced enzymatic activity and thermal stability. Homology modelling revealed that in these mutants some critical stabilizing interactions could no longer occur. The possible critical role of entrance channel and constriction in specific substrate recognition by eukaryotic OSC is discussed. In cholesterol and ergosterol biosynthesis, the most significant structural alteration occurring along the pathway, that is generation of the steroid nucleus after assembly of the triterpene backbone, is brought about by lanosterol synthase. Lanosterol synthase belongs to the large family of OSCs (oxidosqualene cyclases), eukaryotic enzymes that catalyse the cyclization of 2,3-oxidosqualene into different cyclic compounds: lanosterol alone in non-photosynthetic organisms (fungi and mammals), cycloartenol, precursor of phytosterols and other tetra- and pentacyclic compounds in plants [1]. Prokaryotes possess an enzyme similar to OSCs: SHC (squalene-hopene cyclase) that converts squalene into hopene or diplopterol, pentacyclic precursors of hopanoids [2]. Interestingly, while all the known OSCs only accept 2,3oxidosqualene as substrate, SHC not only accepts its physiological substrate squalene, but also the eukaryotes’ substrate, 2,3-oxidosqualene [3]. Squalene and OSCs catalyse among the most fascinating and complex monoenzymatic reactions (Figure 1). During the process, which is primed by protonation of the substrate, four or five rings are formed, several chiral centres are created, hydride and methyl groups are 1,2 shifted and a proton is Key words: enzyme model, homology modelling, mutagenesis, oxidosqualene cyclase (OSC), squalene-hopene cyclase (SHC), thiol inhibitor. Abbreviations used: OSC, oxidosqualene cyclase; SHC, squalene-hopene cyclase. 1 To whom correspondence should be addressed (email [email protected]). C 2005 Biochemical Society extracted (or a hydroxyl added, as in diplopterol formation) and the cyclic product is released [2]. During the past 50 years, an impressive mass of data has been collected on these enzymes; investigations began with pure chemistry and later used inhibitors, mutagenesis studies and homology modelling [2,4,5]. As for other enzymes however, key clues to understanding the structure–function relationship came from solving the crystal structure, which for this class of lipophilic proteins occurred quite recently. In 1997, the structure of SHC from Alicyclobacillus acidocaldarious was solved at medium resolution [6] and a few years later to 2 Å (1 Å = 10−10 m or 0.1 nm) resolution [7]. More recently, the same protein was co-crystallized with the close squalene analogue 2-azasqualene and the complex was analysed by X-ray diffraction to 2.13 Å [8]. Finally, in 2004, human lanosterol synthase was crystallized and its structure solved by a Hoffmann–La Roche group [9]. Crystallographic analysis revealed the presence of a cavity in these proteins, shaped so as to accept the substrate in a prefolded product-like conformation (Figure 2). Both SHC and OSC are monotopic membrane proteins, since they are shaped so as to submerge into the non-polar part of the phospholipid bilayer from one side of the membrane without protruding through it [7–9]. Structural analysis has also shown that the protruding part of the protein contains a lipophilic channel leading to the active-site cavity. Cavity and channel are separated by a narrow constriction formed by four amino acid residues that Seventh Yeast Lipid Conference Figure 1 Reactions catalysed by (A) prokaryotic SHC and (B) lanosterol synthase Reproduced with permission from Milla, P., Lenhart, A., Grosa, G., Viola, F., Weihofen, W.A., Schulz, G.E. and Balliano, G. c Blackwell (2002) Third-modifying inhibitors for understanding squalene cyclase function. Eur. J. Biochem. 269, 2108–2116 Publishing Ltd. Figure 2 Surface representation of SHC sliced in the middle of the molecule A detergent molecule N,N-dimethyldodecylamine-N-oxide that has been found in a crystal structure of SHC [11] is shown by the ball-and-stick model. The catalytic acid Asp376 and the residues forming the channel constriction are indicated. Reproduced with permission from Milla, P., Lenhart, A., Grosa, G., Viola, F., Weihofen, W.A., Schulz, G.E. and Balliano, G. (2002) Third-modifying inhibitors for understanding c Blackwell squalene cyclase function. Eur. J. Biochem. 269, 2108–2116 Publishing Ltd. appear to block it (Figure 2). Two residues of the constriction, however, belong to a loop that appears to be sufficiently mobile to permit passage of the substrate in its extended conformation [7]. When the hypothesis that the substrate entered through the channel constriction of SHC was first raised, we thought we had a suitable tool to verify it: one of the four amino acid residues forming the narrow constriction is a Cys (Cys435 , SHC numbering), an amino acid residue that is easy to modify and we had previously used some thiol reagents as inhibitors of yeast OSC [10]. A reagent could be used as a sort of obstruction device at the constriction of SHC: the entrance gate hypothesis would be confirmed, if an inhibitory effect occurred. The experiments gave the expected results, especially significant because we studied not only a wildtype recombinant protein, which possesses five Cys residues and no disulphide bridges, but also a pair of mutants obtained by site-directed mutagenesis in the laboratory of Schulz [11]: a single mutant lacking the Cys at the channel constriction and a quadruple mutant bearing only the Cys at the channel constriction. Thiol-reacting agents proved more effective on the quadruple than on the single mutant [11]. SHC has long been the only template structure for building homology models that is able to give important mechanistic insights into oxidosqualene cyclization by eukaryotic OSCs [12]. It was obvious, however, that SHC was only of limited use as a model for unravelling the OSC reaction, because of differences between SHC and OSC in substrate, high-energy intermediates and products. The appearance of the SHC structure triggered the hunt for the first eukaryotic cyclase (OSC) structure and at the end of 2004, the news that all OSC searchers were awaiting was announced: the structure of human lanosterol synthase had been solved [9]. The outstanding achievement obtained with human OSC should be considered both as the conclusion of 50 years of history, since it permits the mechanism of the first enzymatic oxidosqualene cyclization to be explored at the molecular level, and as the beginning of new research projects based on predicting the structures of all OSCs cloned thus far, by homology modelling. Concerning the question of substrate access to the active site, the structure of human OSC confirmed the existence of a lipophilic entrance channel and a constriction also in eukaryotic OSC and allowed single residues or sections of the peptide backbone, which can alter their conformation or change their arrangement to permit substrate passage, to be identified [9]. Although the architecture of the lipophilic channel was clear from the crystal structure, its role in C 2005 Biochemical Society 1203 1204 Biochemical Society Transactions (2005) Volume 33, part 5 Figure 3 Alignment of supposed OSCs channel constriction sequences with SHC Alignments were achieved using the MultAlin program (http://prodes.toulouse.inra.fr/multalin/multalin.html). substrate–enzyme interaction is still far from being fully understood. One would wonder, for instance, how specific the channelling of the substrate to the active site through the lipophilic channel and constriction actually is. Is the higher substrate specificity of OSCs (which transform only oxidosqualene) compared with SHCs (which transform both squalene and oxidosqualene) [3] due to the ability of the lipophilic channel of OSCs to reject the squalene nonepoxide before entrance into the active-site cavity, or does the rejection occur after entrance? Several findings suggest that selection occurs outside the active site. For instance, if an extended conformation is required for the substrate to pass through the narrow constriction [7], it might be thought that the substrate passes the constriction and enters the active-site cavity with its epoxide edge turned towards the protonating apparatus located at the top of the active-site cavity. In OSC, the active-site cavity appears to be more spatially restricted than that in SHC [13]; in this case, it might not be easy for the substrate to turn within the active site. If this were the case, the channelling of properly oriented substrate would undoubtedly be a task for the lipophilic channel and channel constriction. Furthermore, metabolic reasons suggest that selection of substrate preferably occurs outside the active site, to reduce possible competitive inhibition of OSC by the close substrate analogue squalene. We approached the problem of the role of the constriction region and lipophilic channel in OSCs through inhibition experiments, site-directed mutagenesis and homology modelling. We planned initially to prepare mutants of lanosterol synthase from Saccharomyces cerevisiae bearing Cys at the channel constriction in order to repeat the labelling experiments with thiol reagents, as we had previously done with SHC [11]. Thus a suitable control mutant had to be prepared. All eukaryotic OSCs possess a critical Cys residue inside the active site, belonging to the highly conserved catalytic motif DCTAE (455–459) [14,15]. We thus needed a control protein lacking the active-site Cys residue, to avoid possible competition for thiol-reacting agents between this residue and the Cys residues that we planned to insert at the constriction. Starting from this control mutant, we prepared channel constriction mutants. Positions where Cys residues were inserted were selected on the basis of both sequence alignment (Figure 3) and homology modelling. Mutants were treated with thiol-reacting agents of different shapes and sizes and their possible obstructing effect was evaluated to verify the role of the constriction as a gate for substrate passage. As C 2005 Biochemical Society expected, Cys-bearing mutants proved to be more susceptible to thiol-reacting reagents than the wild-type. One of the Cys-bearing mutants proved to be less active and less stable than the control enzyme, thus indicating that the site-directed mutagenesis substitution had touched a single critical position of the protein [16]. The homology model revealed that a section of the entrance constriction bearing the critical position is crucially linked via hydrogen bonds to some distant (in sequence) parts of the enzyme. Additional substitution at this position further confirmed its critical role and smoothed the way for a more extensive mutagenesis study. The results obtained with both mutagenesis and inhibition experiments suggest that the entrance channel and the channel constriction could play a critical role in specific substrate recognition by OSC. Much biochemical investigation will be required to confirm the proposed role: novel mutants will have to be tested for their catalytic activity and thermal stability and important information may also be obtained by determining the mutants’ susceptibility to known sitedirected inhibitors such as 2-azasqualene [8] and RO 48-8071 [17,18], and by studying the possible competitive effect of squalene. A challenge raised by the use of human OSC as a template should be the comparison, through homologymodelling studies, of the three-dimensional structures of all the cloned OSCs. Comparison of different entrance channels could clarify the molecular basis of their common ability to specifically recognize the substrate. This work was supported by the Ministero dell’Istruzione, Università e Ricerca (MIUR) (ex 60%). Thanks are due to Professor G. Grosa (Università del Piemonte Orientale ‘Amedeo Avogadro’, Novara, Italy) for supplying thiol-reacting agents and to Professor G.E. Schulz (Universität Freiburg, Germany) for providing the original picture of Figure 2 [11]. References 1 Nes, W.D. (1990) Recent Adv. Phytochem. 24, 283–327 2 Wendt, K.U., Schulz, G.E., Corey, E.J. and Liu, D.R. (2000) Angew. Chem. Int. 39, 2812–2833 3 Abe, I. and Rohmer, M. (1994) J. Chem. Soc. Perkin Trans. 1, 783–791 4 Woodward, R.B. and Bloch, K. (1953) J. Am. Chem. Soc. 75, 2023–2024 5 Abe, I., Rohmer, M. and Prestwich, G.D. (1993) Chem. Rev. 90, 2189–2206 6 Wendt, K.U., Poralla, K. and Schulz, G.E. (1997) Science 277, 1811–1815 7 Wendt, K.U., Lenhart, A. and Schulz, G.E. (1999) J. Mol. Biol. 286, 175–187 Seventh Yeast Lipid Conference 8 Reinert, D.J., Balliano, G. and Schulz, G.E. (2004) Chem. Biol. 11, 121–126 9 Thoma, R., Schulz-Gasch, T., D’Arcy, B., Benz, J., Aebi, J., Dehmlow, H., Hennig, M., Stihle, M. and Ruf, A. (2004) Nature (London) 432, 118–122 10 Balliano, G., Grosa, G., Milla, P., Viola, F. and Cattel, L. (1993) Lipids 28, 903–906 11 Milla, P., Lenhart, A., Grosa, G., Viola, F., Weihofen, W.A., Schulz, G.E. and Balliano, G. (2002) Eur. J. Biochem. 269, 2108–2116 12 Schulz-Gasch, T. and Sthal, M. (2003) J. Comput. Chem. 24, 741–753 13 Dehmlow, H., Aebi, J.D., Jolidon, S., Ji, J.H., von der Marck, E.M., Himber, J. and Morand, O. (2003) J. Med. Chem. 46, 3354–3370 14 Corey, E.J., Matsuda, S.P.T. and Bartel, B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2211–2215 15 Shi, Z., Buntel, C. and Griffin, J.H. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7370–7374 16 Oliaro-Bosso, S., Schulz-Gasch, T., Balliano, G. and Viola, F. (2005) ChemBioChem, in the press 17 Morand, O.H., Aebi, J.D., Dehmlow, H., Ji, J.H., Gains, N., Langsfeld, H. and Himber, J. (1997) J. Lipid Res. 38, 373–390 18 Lenhart, A., Reinert, D.J., Aebi, J.D., Dehmlow, H., Morand, O.H. and Schulz, G.E. (2003) J. Med. Chem. 46, 2083–2092 Received 1 June 2005 C 2005 Biochemical Society 1205