Hopene Cyclase Research Articles

Squalene-hopene cyclase: insight into the role of the methyl group on the squalene backbone upon the polycyclization cascade. Enzymatic cyclization products of squalene analogs lacking a 26-methyl group and possessing a methyl group at C7 or C11.

To provide deep insight into the polycyclization reaction of squalene, some analogs were synthesized and incubated with the cell-free homogenates of the recombinant Escherichia coli encoding the wild-type squalene cyclase. The presence of C6-Me leads to an efficient polycyclization cascade. Substitution of the C14-H and the C18-H with a methyl group halted the polycylization reaction at the tricyclic ring stage having a 6/6/6-fused ring system and the tetracycle with a 6/6/6/6-fused ring, respectively, both of which were produced according to a Markovnikov closure. Replacement of the C7-H and the C11-H with a methyl group led to no cyclization. These results, in conjunction with our previous reports, indicated that the methyl positions are important for bringing to completion of the normal polycylization reaction and further demonstrated that the precise steric bulk size at the methyl positions of squalene is critical to the correct folding and the strong binding of the substrate to the squalene cyclase.

Binding structures and potencies of oxidosqualene cyclase inhibitors with the homologous squalene-hopene cyclase.

The binding structures of 11 human oxidosqualene cyclase inhibitors designed as cholesterol-lowering agents were determined for the squalene-hopene cyclase from Alicyclobacillus acidocaldarius, which is the only structurally known homologue of the human enzyme. The complexes were produced by cocrystallization, and the structures were elucidated by X-ray diffraction analyses. All inhibitors were bound in the large active center cavity. The detailed binding structures are presented and discussed in the light of the IC50 values of these 11 as well as 17 other inhibitors. They provide a consistent picture for the inhibition of the bacterial enzyme and can be used to adjust and improve homology models of the human enzyme. The detailed active center structures of the two enzymes are too different to show an IC50 correlation.

Enzymatic formation of an unnatural hexacyclic C35 polyprenoid by bacterial squalene cyclase.

A C35 polyprene in which a farnesyl C15 unit is connected in a head-to-head fashion to a geranylgeranyl C20 unit was enzymatically converted to an unnatural hexacyclic polyprenoid by squalene:hopene cyclase from Alicyclobacillus acidocaldarius. This is the first demonstration of the remarkable ability of the squalene cyclizing enzyme to perform construction of unnatural hexacyclic skeleton. The cyclization of the C35 polyprene was initiated by a proton attack on the terminal double bond of the C15 unit and proceeded without rearrangement of carbon and hydrogen. The substrate should be folded in chair-chair-chair-chair-boat-boat conformation to achieve the stereochemistry of the cyclization product.

Importance of the Methyl Group at C(10) of Squalene for Hopene Biosynthesis and Novel Carbocyclic Skeletons with 6/5 + 5/5 + (6) Ring System(s)

[reaction: see text] Incubation of (6E,10E,14E,18E)-2,6,10,19,23-pentamethyl-tetracosa-2,6,10,14,18,22-hexaene with Alicyclobacillus acidocaldarius hopene cyclase afforded four products having two types of carbocyclic skeletons, i.e., two hopane products and two products having an unprecedented carbocyclic skeleton of 6/5 + 5/5 +6 pentacyclic and 6/5 + 5/5 tetracyclic ring systems. The former two hopane skeletons were formed from the bioconversion of C15-desmethylsqualene and the latter two skeletons from that of C10-desmethylsqualene.

ChemInform Abstract: Squalene—Hopene Cyclase: Catalytic Mechanism and Substrate Recognition

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Squalene-hopene cyclase: catalytic mechanism and substrate recognition.

Rapid progress on the catalytic mechanism and substrate recognition by squalene-hopene cyclase, which has occurred only in the last several years, is reported. A series of site-directed mutation experiments and some squalene analogues have provided deep insight into the polycyclization mechanism and catalytic sites in conjunction with the information from X-ray crystal data.

Bicyclic triterpenes as new main products of squalene-hopene cyclase by mutation at conserved tyrosine residues.

The catalytic cavity of the Alicyclobacillus acidocaldarius squalene-hopene cyclase is predominantly lined by aromatic amino acids. In mutant cyclases, the four tyrosine residues in the catalytic cavity were replaced by different amino acids. The mutants showed significant differences in catalytic behavior compared to the wild-type and to each other. Mutants Y609L, Y609C and Y609S produced the bicyclic main product gamma-polypodatetraene, while Y495L and Y612L showed a wild-type product pattern and produced hopene as the main product. Altered product patterns were also found with Y420 mutations.

Vinyl sulfide derivatives of truncated oxidosqualene as selective inhibitors of oxidosqualene and squalene-hopene cyclases.

Various vinyl sulfide and ketene dithioacetal derivatives of truncated 2,3-oxidosqualene were developed. These compounds, having the reactive functions at positions C-2, C-15 and C-19 of the squalene skeleton, were studied as inhibitors of pig liver and Saccharomyces cerevisiae oxidosqualene cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene hopene cyclase (SHC) (EC 5.4.99.-). They contain one or two sulfur atoms in alpha-skeletal position to carbons considered to be cationic during enzymatic cyclization of the substrate and should strongly interact with enzyme nucleophiles of the active site. Most of the new compounds are inhibitors of the OSC and of SHC, with various degrees of selectivity. The methylthiovinyl derivative, having the reactive group at position 19, was the most potent and selective inhibitor of the series toward S. cerevisiae OSC, with a concentration inhibiting 500% of the activity of 50 nM, while toward the animal enzyme it was 20 times less potent. These results could offer new insight for the design of antifungal drugs.

Specific production of γ-polypodatetraene or 17-isodammara-20(21),24-diene by squalene–hopene cyclase mutant

Amino acids lining the catalytic cavity of squalene–hopene cyclase of Alicyclobacillus acidocaldarius were mutated to investigate their catalytic functions. Mutagenesis of Leu607 to Lys in the central part of the cavity resulted in the production of the bicyclic γ-polypodatetraene ( 1 ) as main product, while the mutation of Phe605 to Lys near the deprotonation site of the cavity led mainly to the formation of tetracyclic 17-isodammara-20(21),24-diene ( 2 ).

Site-directed mutagenesis of squalene–hopene cyclase: altered substrate specificity and product distribution

Background: Two regions of squalene–hopene cyclase (SHC) were examined to define roles for motifs posited to be responsible for initiation and termination of the enzyme-catalyzed polyolefinic cyclizations. Specifically, we first examined the triple mutant of the DDTAVV motif, a region deeply buried in the catalytic cavity and thought to be responsible for the initiation of squalene cyclization. Next, four mutants were prepared for Glu45, a residue close to the substrate entrance channel proposed to be involved in the termination of the cyclization of squalene. Results: The D D TA VV motif in SHC was changed to D C TA EA , the corresponding conserved region of eukaryotic oxidosqualene cyclase (OSC), by the triple mutation of D377C/V380E/V381A; selected single mutants were also examined. The triple mutant showed no detectable cyclization of squalene, but effectively cyclized 2,3-oxidosqualene to give mono- and pentacyclic triterpene products. Of the Glu45 mutants, E45A and E45D showed reduced activity, E45Q showed slightly increased activity, and E45K was inactive. A normal yield of pentacyclic products was produced, but the ratio of hopene 2 to hopanol 3 was significantly changed in the less active mutants. Conclusions: Initiation and substrate selectivity may be determined by the interaction of the DDTAVV motif with the isopropylidene of squalene (for SHC) and of the DCTAEA motif with the epoxide of oxidosqualene (for OSC). This is the first report of a substrate switch determined by a central catalytic motif in a triterpenoid cyclase. At the termination of cyclization, the product ratio may be largely controlled by Glu45 at the entrance channel to the active site.

Conserved Tyr residues determine functions of Alicyclobacillus acidocaldarius squalene–hopene cyclase

The catalytic cavity of Alicyclobacillus acidocaldarius squalene–hopene cyclase is mainly lined by aromatic amino acids. In recombinant cyclases, three out of four tyrosine residues (Y) have been mutated to phenylalanine residues (F). The mutant cyclases Y495F and Y612F had less activity than the wild-type cyclase, but a wild-type product pattern. Mutant Y609F had wild-type activity but a drastically altered product pattern with hopene and significant amounts of bicyclic α-polypodatetraene and different tetracyclic triterpenes (dammaradienes and eupha-7,24-diene). The experiments demonstrated that Y495 and Y612 may be involved in the initiation of the cyclization reaction and Y609 in the stabilization and/or positioning of the intermediate carbocations.

Functional analysis of Phe605, a conserved aromatic amino acid in squalene–hopene cyclases

Incubation of squalene with the site-directed mutant F605A of squalene–hopene cyclase from Alicyclobacillus acidocaldarius yielded many triterpenes consisting of the 6/6/5-fused tri-, 6/6/6/5-fused tetra-, and 6/6/6/6/5-fused pentacyclic skeletons, the function of F605 being assignable for facilitating the ring expansion and for stabilizing the hopanyl C22-cation, possibly via cation-π interactions.

Enzymatic cyclization of squalene analogs

Abstract

The binding site for an inhibitor of squalene:hopene cyclase determined using photoaffinity labeling and molecular modeling

The squalene:hopene cyclases (SHCs) are bacterial enzymes that convert squalene into hopanoids, a function analogous to the action of oxidosqualene cyclases (OSCs) in eukaryotic steroid and triterpenoid biosynthesis. We have identified the binding site for a selective, potent, photoactivatable inhibitor of an SHC. SHC from Alicyclobacillus acidocaldarius was specifically labeled by [3H]Ro48-8071, a benzophenone-containing hypocholesteremic drug. Edman degradation of a peptide fragment of covalently modified SHC confirmed that Ala44 was specifically modified. Molecular modeling, using X-ray-derived protein coordinates and a single point constraint for the inhibitor, suggested several geometries by which Ro48-8071 could occupy the active site. A covalent complex of a potent inhibitor with a squalene cyclase has been characterized. The amino acid modification and molecular modeling suggest that Ro48-8071 binds at the junction between the central cavity and substrate entry channel, therefore inhibiting access of the substrate to the active site.

Inhibition Kinetics and Affinity Labeling of Bacterial Squalene:Hopene Cyclase by Thia-Substituted Analogues of 2,3-Oxidosqualene

Five sulfur-containing analogues of 2,3-oxidosqualene (OS) were evaluated as inhibitors of squalene:hopene cyclase (SHC) from Alicyclobacillus acidocaldarius. In these analogues, sulfur replaces carbons at C-6, C-10, C-14, C-18, or C-19 of OS. Each analogue was a submicromolar inhibitor of SHC with IC50 values ranging from 60 to 570 nM. Enzyme inhibition kinetic analysis was performed using homogeneous recombinant A. acidocaldarius SHC. While analogues 9 (S-14, Ki = 109 nM, kinact = 0.058 min-1) and 11 (S-19, Ki = 83 nM, kinact = 0.054 min-1) were time-dependent inhibitors of SHC, analogues 7 (S-6, Ki = 127 nM) and 8 (S-10, Ki = 971 nM) showed no time dependency with SHC. Analogue 10 (S-18) was the most potent inhibitor and showed time-dependent irreversible inhibition (Ki = 31 nM, kinact = 0.071 min-1). Kinetic analysis for the five analogues with purified rat liver OSLC was conducted to compare the vertebrate and prokaryotic enzymes. Affinity labeling experiments, using either [17-3H]10 or [22-3H]10 with crude and with pure recombinant SHC, clearly showed specific labeling. A single major radioactive band at 72 kDa on SDS-PAGE indicated that irreversible covalent modification of SHC had occurred. These results suggest that the presence of sulfur at C-18 of OS can interrupt the cyclization and that an intermediate partially cyclized cation may be captured by a nucleophilic residue of the SHC active site.

Photoaffinity labeling of oxidosqualene cyclase and squalene cyclase by a benzophenone-containing inhibitor.

A new orally active oxidosqualene:lanosterol cyclase (OSLC) inhibitor (Ro48-8071; Morand, O. H. et al. (1997) J. Lipid Res. 38, 373-390) showed potent noncompetitive inhibition of bacterial squalene:hopene cyclase (SHC) from Alicyclobacillus acidocaldarius (IC50 = 9.0 nM, KI = 6.6 nM) and OSLC (IC50 = 40 nM, KI = 22 nM for homogeneous rat liver OSLC). A tritium-labeled isotopomer (18.8 Ci/mmol) of this nonterpenoid inhibitor, which possesses a benzophenone (BP) photophore, was chemically synthesized as a photoaffinity label. Specific, efficient covalent modification of both OSLC and SHC enzymes was observed after UV irradiation at 360 nm. Labeling of both OSLC and SHC by [3H]Ro48-8071 was competitively displaced by coincubation with a 1000-fold molar excess of 18-thia-2, 3-oxidosqualene or the nonterpenoid inhibitor BIBX79. Displacement of labeling of OSLC was also achieved with the suicide substrate (3S)-29-methylidene-2,3-oxidosqualene. Thus, the nonsubstrate Ro48-8071 and both terpenoid and nonterpenoid inhibitors of these enzymes appear to share a common binding site.

Squalene–hopene cyclase from Methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids

We report the cloning and characterisation of the Methylococcus capsulatus shc gene, which encodes the squalene–hopene cyclase (SHC). This enzyme catalyses the complex cyclization of squalene to the pentacyclic triterpene skeleton of hopanoids and represents the key reaction in this biosynthesis. Using a combination of PCR amplification and DNA hybridization, two overlapping 2.6 kb PstI and 3.3 kb SalI DNA fragments were cloned bearing a 1962 bp open reading frame encoding a 74 kDa protein with 654 amino acids and a predicted isoelectric point at about pH 6.3. The deduced amino acid sequence of the M. capsulatus shc gene showed significant similarity to known prokaryotic SHCs and to a lesser degree to the related eukaryotic oxidosqualene cyclases (OSCs). Like other triterpene cyclases, the M. capsulatus SHC contains seven so-called QW-motifs as well as an aspartate-rich domain. The recombinant M. capsulatus SHC was expressed in Escherichia coli and in vitro activity of the recombinant cyclase was demonstrated using crude cell-free lysate or solubilized membrane preparation. The cyclization products hop-22-ene and hopan-22-ol (diplopterol) were identified by GC and GC-MS.

Cyclization of (3S)29-Methylidene-2,3-oxidosqualene by Bacterial Squalene:Hopene Cyclase: Irreversible Enzyme Inactivation and Isolation of an Unnatural Dammarenoid

Cyclization of (3<i>S</i>)29-Methylidene-2,3-oxidosqualene by Bacterial Squalene:Hopene Cyclase: Irreversible Enzyme Inactivation and Isolation of an Unnatural Dammarenoid