Oxidosqualene-lanosterol cyclase (OSC)

The formation of lanosterol, the required precursor of cholesterol in vertebrates and ergosterol in fungi, is catalyzed by oxidosqualene-lanosterol cyclase (or lanosterol synthase), the protein encoded from ERG7 gene (EC 5.4.99.7). The oxidosqualene cyclase gene was initially isolated from C. albicans by complementation of an ERG7 mutation in S.

cerevisiae.^[29] This ERG7 gene contains an open reading frame encoding a 731-amino acid, 83kDa protein.

In order to understand the structure-function relationships of oxidosqualene cyclase-catalyzed reactions, both chemical and molecular biological studies have significantly contributed mechanistic insights into the oxidosqualene cyclase-catalyzed cyclization/rearrangement cascade. For example, cloning, sequencing, and site-directed mutagenesis studies of putative oxidosqualene cyclase genes have facilitated understanding of cyclase evolution and analysis of product diversity determining factors, perhaps the single most remarkable feature of cyclase enzymes.

Detailed studies with nonnatural substrates have established the overall pathways of the enzymatic reactions involving: 1) binding of the polyolefinic substrate in a pre-folded conformation, 2) initiation of the reaction by protonation of a double bond (squalene) or an epoxide (2,3-oxidosqualene), 3) ring formation, 4) skeletal rearrangement by 1,2-methyl and hydride shifts, 5) termination by deprotonation or addition of water.^[30]

Crystallization and structural characterization of the membrane protein SHC from Alicyclobacillus acidocaldarius have provided a detailed model for determination of the

substrate entrance channel and catalytically important active-site residues.^{[2, 31-32]}

In 2004, Thoma et al. have succeeded in determining the long-awaited structure of human OSC in complex with the reaction product lanosterol and it provided an important additional snapshot of the triterpene polycyclization cascades (Fig. 1.8a).^[33]

Figure 1.8 Crystal structure of human OSC (a) Ribbon diagram of human OSC. The C and N termini and several sequence positions are labeled. The inner barrel helices are colored yellow. The bound inhibitor, Ro48-8071 (black), indicates the location of the active site. (b) The orientation of OSC relative to one leaflet of the membrane, Ro 48-8071 bind in the certral active-site cavity.^[33]

Base on the observation of crystal structure of monotopic membrane protein human OSC, the membrane-inserted surface consists of a plateau in 25Å diameter and a channel that leads to the active-site cavity. This channel is supposed to allow the substrate oxidosqualene to enter the hydrophobic active site but a constriction site separates it from the active-site cavity. Either the sidechain conformation change in the residues Tyr237, Cys233, and Ile524, or the strained loops rearrangement from 516-524 or 697-699 would lead the substrate passing the constricted site of the channel then enter into the enzyme active site. (Fig. 1.8b).

a b

Cyclization mechanism:

The catalytic mechanism for the cyclization reaction of (3S)-2,3-oxidosqualene to lanosterol involved several steps and a series of discrete conformationally rigid, partially cyclized carbocationic intermediates. The formation of lanosterol is initiated in the pre-chair-boat-chair conformation of 2,3-oxidosqualene from an initial protonation of the epoxide moiety by Asp455 ( in human OSC numbering). Protonation of this epoxide ring would trigger a cascade of ring-forming reactions to the protosterol cation formation.

Skeletal rearrangement of this intermediate cation through a series of 1,2-hydride and 1,2-methyl group would shift the cation to the C-8 (lanosterol numbering) cation production, and a final deprotonation step would lead to the product lanosterol generation (Fig. 1.9).

+ Above molecular plane

Below molecular plane

Initiation

In early work to predict the catalytically important residues, Corey et al. set a series of alanine scanning site-directed mutations of the highly conserved amino acids from S.

cerevisiae ERG7. Complementation experiment results showed that His146, H234, and

Asp456 were the catalytically essential residues. From this, a model was proposed that the protonated His146 would increases the acidity of Asp456 which acts as the proton donor for initiating cyclization.^[34-35] In parallel, the X-ray structure of humanOSC also showed that Asp455 (Asp456 of S. cerevisiae ERG7) is hydrogen-bonding to Cys456 and Cys533, and it contributes to the required acidity for the protonation of epoxide. And then Asp455 can be reprotonated from a H-bonding network of bulk solvent (Fig. 1.10).^[30,33] Based on experimental and theoretical studies it is widely accepted that initiation reaction was happening via the protonation and the following concerted A ring was also carried out.^[36-40]

Figure 1.10 H. sapiens OSC (turquoise) with lanosterol (yellow): polar residues around catalytic acid Asp455, polar interactions, and hydrogen-bonding network.

Ring formation

In early studies about the B-ring formation, Matsuda et al. found that Val454 is strictly conserved in the S. cerviase OSC and the corresponding residue Ile481 in A.

thaliana cycloartenol synthase was also considered as a catalytically important residue. A

series of Val454 mutants were substituted as the hydrophobic residues in decreasing the amino acid side chain size of steric bulk from isopropyl to methyl or hydrogen (Phe, Leu, Ile, Ala and Gly). The Ala and Gly mutants allow the monocyclic products formation. It was suggested that steric bulk contributed from the valine side chain might participate in prefolding of the oxidosqualene especially in the Δ¹⁰ olefin condensation in the B-ring formation.^[41] On the basis of X-ray crystallographic analysis of human OSC structure, Thomal et al. suggested that highly conserved aromatic residues Trp387, Phe444, and Trp581 (which corresponds to Trp390, Phe445, and Trp 583 of S. cerevisiae ERG7) stabilize the intermediate tertiary cation at C-6 and C-10 through cation-π interaction.

However, generation of truncated tricyclic and altered deprotonation products from the Phe445 site-directed mutagenesis from S. cerevisiae provides an evidence that Phe445 might affect the cationic stabilization at the C-14 position for C-ring formation and also at C-8/C-9 position for final deprotonation.^[42]

Moreover, the induction of the energetically unfavorable boat conformation of the B-ring is thought to be generated via the optimally positioned Tyr98 (Tyr99 of S. cerevisiae ERG7) pushing the methyl group at C-8 (lanosterol numbering) below the molecular plane of oxidosqualene (Fig. 1.11).

Figure 1.11 Trp387, Phe444 and Trp581 are able to stabilize the cyclization intermediates cation C-6 and C-10 through cation-π interaction. Catalytic Asp455 is activated by Cys456 and Cys533. The Tyr 98 side chain contributes the energetically unfavorable boat conformation of B-ring.

The concept of C-ring closure with subsequent ring expansion was initially suggested by Corey et al. based on the finding that the cyclization of 20-oxaoxidosqualene by the OSC of S. cerevisiae yields not only the expected 6-6-6-5 product but 6-6-5 fused-ring product additionally (Fig. 1.12).[2, 30, 43]

O

Figure 1.12 Substrate analogues and products catalyzed by S. cerevisiae OSC which are suggestive of a five-membered C-ring intermediate.

Observations of numerous partially cyclized 6-6-5 and 6-6-6-5 side products arising from the substrate-analogue studies have been considered to support the relevance of the C-14 cation intermediate and also provided further evidence for a five-membered C-ring closure (Markovnikov) followed by subsequent ring expansion. Steric pressure through the enzyme, however, might only play a secondary role for the prefolded C-ring conformation

formation, because the energetically favorable chair conformation is required for the C-ring formation.^[7] Computational studies also provides further support for the 6-6-5 to 6-6-6 rearrangement pathway.^[37] In addition, Thoma et al. showed that side chains of His232 and Phe696 (His234 and Phe699 in S. cerevisiae ERG7) are situated in well position for stabilizing the anti-Markovnikov secondary cation at C-14 with π–cation interactions during C-ring formation through the X-ray crystallographic analysis of human OSC protein.

Rearrangement and deprotonation

Due to the lack of an aromatic residue like Trp169 in SHC for stabilizing the long-lived secondary cation at C-17 for the six-membered E-ring cyclization of hopene, the end of OSC cyclization cascade is stopped at the formation of the five-membered D- ring.

Skeletal rearrangement through 1,2-shift of hydride and methyl substituents (Fig. 1.9) convert the protosterol cation to lanosterol C-8 or C-9 cation. It was confirmed that the high π–electron density in the enzyme active site could stabilize the positive charge intermediate during the rearrangement. Thus, the enzyme’s role in skeletal rearrangement is thought to shift equilibrium between the protosteryl cation toward carbocations at the C-8 and C-9 position of lanosteryl cation.^{[7, 33]}

After skeletal rearrangement to C-8 and C-9 lanosterol cation the cyclization is terminated by proton removement. In the deprotonation step, OSC displays the specific catalytic selectivity for lanosterol synthesis which is the most thermodynamic stable deprotonation product (Saytzeff product) with a tetrasubstituted double bond. Thomal’s group confirmed that His 232 (H234 in S. cerevisiae ERG7) is the only basic residue in the proximity to the termination site,[2,7, 34-35] and His232 would also form a hydrogen bond with the hydroxy group of Tyr503 (Tyr510 in S. cerevisiae ERG7 ), which is in a better position to accept the proton from C-9 of the lanosterol cation than His232 itself. The

interpretation of His232 and Tyr 503 as the catalytic base dyad in OSC was supported by site-directed mutagenesis data (Fig. 1.13): 1) The site-saturated mutagenesis experiments of His234 residue in S. cerevisiae ERG7 generated multiple triterpene products including protosta-20,24-dien-3β-ol, protosta-12,24-diene-3β-ol and parkeol from various ERG7^His234x mutants^[44-45] and 2) the ERG7^Tyr510Ala mutant in S. cerevisiae formed parkeol.^[46,47] By the same token, the functional analysis of Trp232 in S. cerevisiae ERG7 illustrated that Trp232 might play a catalytic role in the influence of rearrangement process and determination of deprotonation position but does not intervene in the cyclizaiotn steps (Fig. 1.13).^[48]

ERG7^Tyr510X ERG7^Tyr510X

ERG7^His234X

ERG7^His234X

ERG7^His234X

ERG7^His234X

ERG7^His234X ERG7^His234X

ERG7^Trp232X

ERG7^Trp232X ERG7^Trp232X

Figure 1.13 The products diversity from site-saturated mutant of His234, Tyr510 and Trp232.