Squalene-hopene cyclase

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Squalene-hopene cyclase
2sqc.png
The crystallographic structure of the squalene-hopene cyclase dimer, with the membrane position indicated in blue, the two monomers in green and pink, and a substrate mimetic in the central cavity in yellow. [1]
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EC no. 5.4.99.17
CAS no. 76600-69-6
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Squalene-hopene cyclase (SHC) (EC 5.4.99.17) or hopan-22-ol hydro-lyase is an enzyme in the terpene cyclase/mutase family. It catalyzes the interconversion of squalene into a pentacyclic triterpenes, hopene and hopanol. [2] [3] [4] [5] [6] This enzyme catalyses the following chemical reactions.

Contents

squalene hop-22(29)-ene
squalene + H2O hopan-22-ol

SHC is important because its products, hopanoids, are very much like sterols in eukaryotes in that they condense lipid membranes and reduce permeability. In fact, SHC and sterol-producing enzymes (oxidosqualene cyclase) are evolutionarily related to each other. [7] Hopanoids are inferred to provide stability in the face of high temperatures and extreme acidity due to the rigid ring structure. [8] Indeed, up-regulation of SHC occurs in certain bacteria in the presence of hot or acidic environments. [9] [10] SHC is found mostly in bacteria, but some eukaryotes, such as fungi and land plants, are also known to possess the enzyme.

Chemical structure of hopene Hopene.svg
Chemical structure of hopene

Introduction

SHC is found in a large number of bacteria [7] but is most readily isolated from the thermophilic bacterium Alicyclobacillus acidocaldarius. [11]

Space-filling model of the squalene molecule Squalene-from-xtal-3D-vdW-A.png
Space-filling model of the squalene molecule

SHC does not require molecular oxygen for its reaction and is thought to be an evolutionary progenitor of oxygen-dependent oxidosqualene cyclase (OSC), which produces tetracyclic sterols. OSC is a eukaryotic analog of SHC and requires molecular oxygen for its catalysis. This may suggest a later evolution of OSC relative to SHC, when the atmosphere began accumulating oxygen, [12] although the distribution of SHC is also limited mostly to aerobic species.

Structure

Squalene-hopene cyclase is a membrane-associated 70-75kDa protein composed of 631 amino acids and seven PTFB repeats. It exists as a monotopic homodimer. [1]

Mechanism

The formation of the hopene skeleton is one of the most complex single-step reactions in biochemistry. [13] In a single step, 13 covalent bonds are broken or formed, 9 chiral centers are established, and 5 rings are produced. [14] Squalene–hopene cyclase catalyses the conversion of the acyclic molecule of squalene into the pentacyclic triterpenes of hopene and hopanol. These products appear in the ratio 5:1. Hopene synthesis begins with binding squalene in an all pre-chair conformation and is followed by the formation of five C-C bonds. [15] These sequential ring-forming reaction steps are initiated by an electrophilic attack of an acidic proton on one of the two terminal double bonds. The polycyclic formation is completed when a proton is eliminated from the alternative terminal methyl group of squalene via acceptance by a water molecule. [5] This base is known as the front water. Other water molecules work to enhance polarization (back waters) and construct hydrogen bonds between seven residues—T41, E45, E93, R127, Q262, W133 and Y267. Front water also plays a role in determining the end product. If it stores the proton generated from either Methyl group 29 or 30 to form hopene. However, hopanol is produced in lesser quantities if instead of accepting the proton, water contributes a hydroxyl to the C-22 cation of the A-ring. [16]

Suggested active residues in squalene-hopene cyclase Active site and Asp.png
Suggested active residues in squalene-hopene cyclase

During the formation of rings A through D, there is very little conformational change. The reaction therefore requires no intermediate and can take place in one step. However, ring E formation is hindered by an entropic barrier, which may explain its absence in the tetracyclic steroids. [5]

Active site

The SHC active site is located in a central cavity within the region of the protein adjacent to the membrane, and is accessed by the substrate via a non-polar channel. [17] The active site is notably surrounded by aromatic residues forming a cavity that comfortably fits the squalene molecule when folded into a productive conformation. The catalytic mechanism uses coupled aspartate and histidine residues to initiate the cyclization reaction by protonating at C3 and deprotonating at C29, proceeding through a discrete series of carbocation intermediates. [1] [18] The enzyme can be inactivated by mutation of catalytic aspartates. [19]

Thermodynamics

This enzyme is unusually exothermic with an energy release of 40-50kcal/mol, well beyond the protein stabilization energy. This is thought to melt a lipid side channel through which the bulky product exits. In order to maintain its structural integrity, some scientists believe that the enzyme’s 7-8 non-tandem repeat QW motifs (Q is glutamine and W is tryptophan) that connect numerous surface α helices tighten the protein structure and prevent denaturing. [1]

Numerous tightly linked surface helices Surface helices.png
Numerous tightly linked surface helices

Related Research Articles

<span class="mw-page-title-main">Steroid</span> Any organic compound having sterane as a core structure

A steroid is a biologically active organic compound with four rings arranged in a specific molecular configuration. Steroids have two principal biological functions: as important components of cell membranes that alter membrane fluidity; and as signaling molecules. Hundreds of steroids are found in plants, animals and fungi. All steroids are manufactured in cells from the sterols lanosterol (opisthokonts) or cycloartenol (plants). Lanosterol and cycloartenol are derived from the cyclization of the triterpene squalene.

<span class="mw-page-title-main">Squalene</span> Chemical compound

Squalene is an organic compound. It is a triterpenoid with the formula C30H50. It is a colourless oil, although impure samples appear yellow. It was originally obtained from shark liver oil (hence its name, as Squalus is a genus of sharks). An estimated 12% of bodily squalene in humans is found in sebum. Squalene has a role in topical skin lubrication and protection.

<span class="mw-page-title-main">Hopanoids</span> Class of chemical compounds

Hopanoids are a diverse subclass of triterpenoids with the same hydrocarbon skeleton as the compound hopane. This group of pentacyclic molecules therefore refers to simple hopenes, hopanols and hopanes, but also to extensively functionalized derivatives such as bacteriohopanepolyols (BHPs) and hopanoids covalently attached to lipid A.

<span class="mw-page-title-main">Lanosterol</span> Chemical compound

Lanosterol is a tetracyclic triterpenoid and is the compound from which all animal and fungal steroids are derived. By contrast plant steroids are produced via cycloartenol.

<span class="mw-page-title-main">Sterane</span> Class of tetracyclic compounds derived from steroids

Sterane (cyclopentanoperhydrophenanthrenes) compounds are a class of tetracyclic triterpanes derived from steroids or sterols via diagenetic and catagenetic degradation, such as hydrogenation. Steranes are detected in sediments and sedimentary rocks in nature. Steranes have an androstane skeleton with a side chain at carbon C-17. The sterane structure constitutes the core of all sterols. Steranes are widely used as biomarkers for the presence of eukaryotes in past ecosystems because steroids are nearly exclusively produced by eukaryotes. In particular, cholestanes are diagenetic products of cholesterol in animals, while stigmastanes are diagenetic products of stigmasterols in algae and land plants. However, some bacteria are now known to produce sterols and it is inferred that the ultimate origin of sterol biosynthesis is in bacteria. Sterols are produced via protosterols that are direct cyclization compounds of squalene by the catalysis of oxidosqualene cyclase. All known sterols in eukaryotes are enzymatically extensively modified from protosterols, while organisms that only produce protosterols are not known. The oldest record of modified steranes are in sedimentary rocks deposited ca. 720–820 million years ago. In contrast, diagenetic products of protosterols are widely distributed in older Proterozoic rocks and imply the presence of extinct proto-eukaryotes and/or sterol-producing bacteria before the evolution of crown-group eukaryotes.

<span class="mw-page-title-main">Triterpene</span> Class of chemical compounds

Triterpenes are a class of terpenes composed of six isoprene units with the molecular formula C30H48; they may also be thought of as consisting of three terpene units. Animals, plants and fungi all produce triterpenes, including squalene, the precursor to all steroids.

<span class="mw-page-title-main">Farnesyl-diphosphate farnesyltransferase</span> Class of enzymes

Squalene synthase (SQS) or farnesyl-diphosphate:farnesyl-diphosphate farnesyl transferase is an enzyme localized to the membrane of the endoplasmic reticulum. SQS participates in the isoprenoid biosynthetic pathway, catalyzing a two-step reaction in which two identical molecules of farnesyl pyrophosphate (FPP) are converted into squalene, with the consumption of NADPH. Catalysis by SQS is the first committed step in sterol synthesis, since the squalene produced is converted exclusively into various sterols, such as cholesterol, via a complex, multi-step pathway. SQS belongs to squalene/phytoene synthase family of proteins.

(S)-2,3-Oxidosqualene ((S)-2,3-epoxysqualene) is an intermediate in the synthesis of the cell membrane sterol precursors lanosterol and cycloartenol, as well as saponins. It is formed when squalene is oxidized by the enzyme squalene monooxygenase. 2,3-Oxidosqualene is the substrate of various oxidosqualene cyclases, including lanosterol synthase, which produces lanosterol, a precursor to cholesterol.

<span class="mw-page-title-main">Lanosterol synthase</span> Mammalian protein found in Homo sapiens

Lanosterol synthase is an oxidosqualene cyclase (OSC) enzyme that converts (S)-2,3-oxidosqualene to a protosterol cation and finally to lanosterol. Lanosterol is a key four-ringed intermediate in cholesterol biosynthesis. In humans, lanosterol synthase is encoded by the LSS gene.

<span class="mw-page-title-main">Isopentenyl-diphosphate delta isomerase</span> Class of enzymes

Isopentenyl pyrophosphate isomerase, also known as Isopentenyl-diphosphate delta isomerase, is an isomerase that catalyzes the conversion of the relatively un-reactive isopentenyl pyrophosphate (IPP) to the more-reactive electrophile dimethylallyl pyrophosphate (DMAPP). This isomerization is a key step in the biosynthesis of isoprenoids through the mevalonate pathway and the MEP pathway.

<span class="mw-page-title-main">Prenyltransferase</span>

Prenyltransferases (PTs) are a class of enzymes that transfer allylic prenyl groups to acceptor molecules. Prenyl transferases commonly refer to isoprenyl diphosphate syntheses (IPPSs). Prenyltransferases are a functional category and include several enzyme groups that are evolutionarily independent.

Squalene—hopanol cyclase (EC 4.2.1.129, squalene—hopene cyclase) is an enzyme with systematic name hopan-22-ol hydro-lyase. This enzyme catalyses the following chemical reaction

β-amyrin synthase is an enzyme with systematic name (3S)-2,3-epoxy-2,3-dihydrosqualene mutase . This enzyme catalyses the following chemical reaction

Lupeol synthase is an enzyme with systematic name (3S)-2,3-epoxy-2,3-dihydrosqualene mutase . This enzyme catalyses the following chemical reaction

<span class="mw-page-title-main">Parkeol</span> Chemical compound

Parkeol is a relatively uncommon sterol secondary metabolite found mostly in plants, particularly noted in Butyrospermum parkii. It can be synthesized as a minor product by several oxidosqualene cyclase enzymes, and is the sole product of the enzyme parkeol synthase.

<span class="mw-page-title-main">Oxidosqualene cyclase</span>

Oxidosqualene cyclases (OSC) are enzymes involved in cyclization reactions of 2,3-oxidosqualene to form sterols or triterpenes.

<span class="mw-page-title-main">Taraxasterol</span> Chemical compound

Taraxasterol (anthesterin) is a triterpene derived from the mevalonate pathway and is found in dandelions.

<span class="mw-page-title-main">Tetrahymanol</span> Chemical compound

Tetrahymanol is a gammacerane-type membrane lipid first found in the marine ciliate Tetrahymena pyriformis. It was later found in other ciliates, fungi, ferns, and bacteria. After being deposited in sediments that compress into sedimentary rocks over millions of years, tetrahymanol is dehydroxylated into gammacerane. Gammacerane has been interpreted as a proxy for ancient water column stratification.

<span class="mw-page-title-main">Isoarborinol</span> Chemical compound

Isoarborinol is a triterpenoid ubiquitously produced by angiosperms and is thus considered a biomarker for higher plants. Though no isoarborinol-producing microbe has been identified, isoarborinol is also considered a possible biomarker for marine bacteria, as its diagenetic end product, arborane, has been found in ancient marine sediments that predate the rise of plants. Importantly, isoarborinol may represent the phylogenetic link between hopanols and sterols.

<span class="mw-page-title-main">Diplopterol</span> Chemical compound

Diplopterol is a triterpenoid molecule commonly produced by bacteria, ferns, and a few protozoans. This compound, classified as a member of the hopanoid family, is synthesized from triterpenoid precursor squalene. It is generally believed that hopanoids serve a similar function in bacteria as that of sterols in eukaryotes, which involves modulating membrane fluidity. Diplopterol serves as a useful biomarker for prokaryotic life, along with oxygen content at the time of sediment deposition.

References

  1. 1 2 3 4 Wendt KU, Poralla K, Schulz GE (September 1997). "Structure and function of a squalene cyclase". Science. 277 (5333): 1811–5. doi:10.1126/science.277.5333.1811. PMID   9295270.
  2. ↑ Hoshino T, Sato T (February 2002). "Squalene-hopene cyclase: catalytic mechanism and substrate recognition". Chemical Communications (4): 291–301. doi:10.1039/b108995c. PMID   12120044.
  3. ↑ Hoshino T, Nakano S, Kondo T, Sato T, Miyoshi A (May 2004). "Squalene-hopene cyclase: final deprotonation reaction, conformational analysis for the cyclization of (3R,S)-2,3-oxidosqualene and further evidence for the requirement of an isopropylidene moiety both for initiation of the polycyclization cascade and for the formation of the 5-membered E-ring". Organic & Biomolecular Chemistry. 2 (10): 1456–70. doi:10.1039/b401172d. PMID   15136801.
  4. ↑ Sato T, Kouda M, Hoshino T (March 2004). "Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius". Bioscience, Biotechnology, and Biochemistry. 68 (3): 728–38. doi: 10.1271/bbb.68.728 . PMID   15056909.
  5. 1 2 3 Reinert DJ, Balliano G, Schulz GE (January 2004). "Conversion of squalene to the pentacarbocyclic hopene". Chemistry & Biology. 11 (1): 121–6. doi: 10.1016/j.chembiol.2003.12.013 . PMID   15113001.
  6. ↑ Pearson A, Budin M, Brocks JJ (December 2003). "Phylogenetic and biochemical evidence for sterol synthesis in the bacterium Gemmata obscuriglobus". Proceedings of the National Academy of Sciences of the United States of America. 100 (26): 15352–7. Bibcode:2003PNAS..10015352P. doi: 10.1073/pnas.2536559100 . PMC   307571 . PMID   14660793.
  7. 1 2 Santana-Molina, Carlos; Rivas-Marin, Elena; Rojas, Ana M; Devos, Damien P (2020-07-01). Ursula Battistuzzi, Fabia (ed.). "Origin and Evolution of Polycyclic Triterpene Synthesis". Molecular Biology and Evolution. 37 (7): 1925–1941. doi:10.1093/molbev/msaa054. ISSN   0737-4038. PMC   7306690 . PMID   32125435.
  8. ↑ Kannenberg, E.; Poralla, K. (1999). "Hopanoid biosynthesis and function in bacteria". Naturwissenschaften. 86 (4): 168–176. Bibcode:1999NW.....86..168K. doi:10.1007/s001140050592. S2CID   21596134.
  9. ↑ Ourisson G, Rohmer M, Poralla K (1987). "Prokaryotic hopanoids and other polyterpenoid sterol surrogates". Annual Review of Microbiology. 41: 301–33. doi:10.1146/annurev.mi.41.100187.001505. PMID   3120639.
  10. ↑ Sahm H, Rohmer M, Bringer-Meyer S, Sprenger GA, Welle R (1993). "Biochemistry and physiology of hopanoids in bacteria". Advances in Microbial Physiology. 35: 247–73. doi:10.1016/s0065-2911(08)60100-9. ISBN   9780120277353. PMID   8310881.
  11. ↑ Seckler, B.; Poralla, K. (1986). "Characterization and partial purification of squalene-hopene cyclase from Bacillus acidocaldarius". Biochimica et Biophysica Acta (BBA) - General Subjects. 881 (3): 356–363. doi:10.1016/0304-4165(86)90027-9.
  12. ↑ Rohmer, M.; Bouvier, G.; Ourisson, G. (1979). "Molecular evolution of biomembranes: structural equivalents and phylogenetic precursors of sterols". Proceedings of the National Academy of Sciences. 76 (2): 847–851. Bibcode:1979PNAS...76..847R. doi: 10.1073/pnas.76.2.847 . PMC   383070 . PMID   284408.
  13. ↑ Siedenburg G, Jendrossek D (June 2011). "Squalene-hopene cyclases". Applied and Environmental Microbiology. 77 (12): 3905–15. Bibcode:2011ApEnM..77.3905S. doi:10.1128/aem.00300-11. PMC   3131620 . PMID   21531832.
  14. ↑ Corey EJ, Matsuda SP, Bartel B (December 1993). "Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functional expression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen". Proceedings of the National Academy of Sciences of the United States of America. 90 (24): 11628–32. Bibcode:1993PNAS...9011628C. doi: 10.1073/pnas.90.24.11628 . PMC   48037 . PMID   7505443.
  15. ↑ Zheng YF, Abe I, Prestwich GD (April 1998). "Inhibition kinetics and affinity labeling of bacterial squalene:hopene cyclase by thia-substituted analogues of 2, 3-oxidosqualene". Biochemistry. 37 (17): 5981–7. doi:10.1021/bi9727343. PMID   9558334.
  16. ↑ "BRENDA - Information on EC 4.2.1.129 - squalene-hopanol cyclase".
  17. ↑ Gao Y, Honzatko RB, Peters RJ (October 2012). "Terpenoid synthase structures: a so far incomplete view of complex catalysis". Natural Product Reports. 29 (10): 1153–75. doi:10.1039/C2NP20059G. PMC   3448952 . PMID   22907771.
  18. ↑ Hoshino T, Sato T (February 2002). "Squalene-hopene cyclase: catalytic mechanism and substrate recognition". Chemical Communications (4): 291–301. doi:10.1039/B108995C. PMID   12120044.
  19. ↑ Feil, C.; Sussmuth, R.; Jung, G.; et al. (1996). "Site-directed mutagenesis of putative active-site residues in squalene-hopene cyclase". European Journal of Biochemistry. 242 (1): 51–55. doi: 10.1111/j.1432-1033.1996.0051r.x . PMID   8954152.
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