The hypothesis of oxidosqualene cyclase

The family of oxidosqualene cyclases extensively exists in organisms. These different enzyme systems convert the oxidosqualene, as a substrate, into various polycyclic triterpenes. The relationship between the enzyme structure and cyclization mechanism is extremely interesting and diverse.

Woodward and Bloch first proposed in 1953 that in the cholesterol biosynthesis pathway, lanosterol was produced after the cyclization of squalene, followed by rearrangement.⁴ In 1958 and 1965, Maudgal and Cornforth groups provided evidence for 1.2-methyl and hydride shifts during lanosterol formation by incorporation experiments.^5-6 Corey and van Tamelen showed that 2,3-oxidosqualene is more efficiently incorporated in sterol synthesis than squalene, the demonstrated intermediacy of 2,3-oxidosqualene in lanosterol biosynthesis.^7-8 In addition, van Tamelen also showed that the nonenzymatic cyclization of 2,3-oxidosqualene resulted in truncated cyclization to produce a tricyclic product, suggesting that direct enzymatic control is necessary for the prevention of the chemical tendency in the formation of the five-membered C-ring, and for emergence of the biologically required six-membered C-ring.⁹ In 1975, Barton confirmed that eukaryotic oxidosqualene cyclases accepted only (3S)-, but not (3R)-, enantiomers of 2,3-oxidosqualene as a substrate in the formation of lanosterol, demonstrating the highly substrate-specific property of oxidosqualene cyclases compared to squalene cyclases.¹⁰ Guy Ourisson and his co-workers proposed the possible molecular evolution from the primitive squalene cyclases to oxidosqualene cyclases in higher organisms.¹¹ Furthermore, Corey and Matsuda showed that oxirane cleavage and cyclization of the A-ring are concerted and essential for electrophilic activation of the oxirane function.^12-13

In previous research, oxidosqualene was stable in the neutral condition at room temperature for the whole day. A stronger acid such as trichloroacetic acid is

7

required for epoxide ring activation to promote the cyclization step.¹⁴ Then, many unstable cation intermediates were thus formed in the cyclization process. Two hypotheses that showed how the enzyme worked to stabilize the high-energy cation intermediates were proposed. In 1987, Johnson proposed the “cation-stabilizing auxiliary” model which supposed that the Lewis acid residues on the active site provide a proton for initiation of the epoxide group, and a number of anionic sites in the cyclase enzyme which further led to the cation generation and the formation of the proper ring system. These axial negative charge residues would face toward the transition states or the intermediates for the cation stabilization, and also facilitate the ring formation of B-boat/chair ring. Therefore the B-boat ring could be promoted by the delivery of a point charge to the α-face at pro-C-8, and lowering the activation energy of the boat form rather than that of chair skeleton (Fig. 1.3).^15-16 In addition, Griffin and co-workers proposed the “aromatic hypothesis” model from which the electron-rich aromatic side chain, such as Trp and Tyr, might stabilize the positively charged transition states or high-energy intermediates during the process of cyclization and rearrangement steps (Fig. 1.4).¹⁷ In this hypothesis, the aromatic residues play the role of the anions group, just like that proposed in the “Johnson model”. These cation-π interactions are common features in enzyme-substrate complexes.

8

Figure 1.4 Griffin’s hypothesis model for involvement of electron-rich aromatic side chains from Trp and Tyr residues in the cyclization of oxidosqualene to the protosteryl cation.

Figure 1.3 The proposed enzyme models by Johnson

HO

H

HO H O Enz AH⁺

O Enz AH⁺

Dammarenyl Cation Protosteryl Cation

H

H

H

H

denotes site for delivery of negative point charge

9

1.3.2 Squalene-hopene cyclase (SHC)

Squalene-hopene cyclase (EC 5.4.99.17) is a homodimeric enzyme which is organized in two groups of α-helical domains, which together form a dumbbell-shaped molecule (Figure 1.5). It contains 631 amino acids per subunit, and the molecular masses are about 71.5 kDa each.

In 1997, the X-ray crystal structure of Alicyclobacillus acidocaldarius SHC was first reported at a 2.9 Å resolution, later refined to 2.0 Å resolution, in 1999.^18-19 Moreover, Reinert et al. reported another X-ray crystal structure from which the squalene-hopene cyclase was cocrystalized with 2-azasqualene and its resolution is 2.13 Å in 2004.²⁰ These structures combined with the biological studies, provided a more mechanistic insight into squalene-hopene cyclases and oxidosqualene cyclases.

Squalene-hopene cyclase converts squalene to the pentacyclic hopene skeleton in prokaryotes. It binds squalene in the all-chair conformation, and initiates the cyclization cascade by protonating the terminal double bond. The cyclization reaction produces the 6.6.6.6.5-fused pentacyclic hopanyl C-22 cation, which

Figure 1.5 Crystal structure of A. acidocaldarius squalene-hopene cyclase.

10

undergoes either proton elimination or addition of water to produce a 5:1 mixture of hopene and hopanol without the rearrangement step (Figure 1.6).²¹ However, the bacterial SHCs displayed very low substrate specificity. They can cyclize not only the natural substrate, but both enantiomers of oxidosqualene and regular polyprenols.

1.3.3 Oxidosqualene-lanosterol cyclase (OSC)

Oxidosqualene-lanosterol cyclase (EC 5.4.99.7), also called lanosterol synthase, is widely found in animals and fungi. It converts oxidosqualene into the tetracyclic product, lanosterol. Lanosterol is the precursor of ergosterol in fungi and cholesterol in animals, both them are important components of the cell membrane.

In Saccharomyces cerevisiae, cyclases are encoded from erg7, as a membrane protein. The protein contains 731 amino acids and the molecular mass is approximately 83 kDa. Because of the difficulty in purifying membrane proteins, the crystal structure of S. c oxidosqualene-lanosterol cyclases (SceERG7) enzyme has not been possible until recently. In order to understand the structure-function relationships of oxidosqualene cyclase-catalyzed reactions in depth, site directed /

4

Figure 1.6 The cyclization process of squalene-hopene cyclase.

11

saturated mutagenesis, coupled with genetic selection and products analysis, for illustrating the functional importance of these mutated residues, participating in the carbon cationic intermediates stabilization of the reaction cascade were carried out.

The mechanism of oxidosqualene- lanosterol cyclase includes substrate pre-folding, an epoxide ring opening, four rings cyclization, hydride and methyl groups migration, and a final deprotonative termination (Fig. 1.7). In the detailed aspect, the enzyme catalyzed conversion of (3S)-2,3-oxidosqualene into a pre-folded chair-boat-chair conformation. Then, a residue Asp456 (SceERG7) provided a proton to start the reaction, permitting the formation of A-ring. In addition, some researches showed that His146 assists in the formation of hydrogen bond with Asp456, and it enhances the acidity of Asp456, to induce the epoxide ring opening (Fig.1.8).²²

Figure 1.8 The proposed model for oxirane ring opening and cyclization initiation.

Figure 1.7 The mechanism of

conversion of oxidosqualene

into lanosterol.

12

The formation of the B-boat ring probably occurs very rapidly when the positive charge appears on the C-6 of the A-ring. The closure of the C-ring is puzzling because the direct formation of the six-membered C-ring would represent the anti-Markovnikov form. In 1995, E. J. Corey and his co-workers first proposed the five-membered C-ring Markovnikov cyclization against the direct formation of a six-membered C-ring. By using oxidosqualene analogues, 20-oxaoxidosqualene was reacted with oxidosqualene cyclase, and the resulting products suggested that there was a five-membered C-ring intermediate appearing through an intramolecular reaction (Fig. 1.9).²³ Moreover, incubation of additional substrate analogues within oxidosqualene cyclases produced 6-6-5 cyclization products, providing more evidence for a five-membered C-ring closure followed by a ring expansion.²⁴ In the theoretical calculation, Hess indicated that the 6-6-5-tricyclic cation was the first intermediate during the formation of the protosteryl cation, and five-membered C-ring expansion and formation of the D-ring were concerted (Fig. 1.10).²⁵ In comparison, for the SHC study, Gao showed no and fewer intermediates of tricyclic and tetracyclic cations, suggesting that the formation of C, D and E-ring were

Figure 1.9 Substrate analogue and their product derivatives during the oxidosqualene cyclase cyclization which are suggestive of

five-membered C-ring intermediates.

13

Early studies of the sterol biosynthesis assumed that the conversion of oxidosqualene into lanosterol proceed via a protosteryl cation where its cationic sidechain at C-17 is α-oriented.²⁷ After a hydride migration of this 17α-protosteryl cation from C-17 to C-20, an un-natured 20S configuration, but not the natural 20R skeleton product, was thus generated. Another exception for the mechanistic illustration was suggested to explaining the backbone rotation at C20, where a 120°

rotation of C17-C20 bond occurs via the hydride migration in order to produce the 20R skeleton of lanosterol, whereas only 60° rotation is required to produce the unnatural 20S configuration (Fig. 1.11).²⁸ In recent years, it had been proposed that the 17α-protosteryl cation may not be the intermediate. In 1992, Corey proposed the evidence that stereochemistry at C-17 of the protosteryl cation prefers a β- rather than an α- orientation by the enzyme-catalyzed cyclization of 20-oxaoxidosqualene

(Fig. 1.12). This result overthrows the assumption that the large rotation around

C17-C20 bond occurs prior to the actual rearrangement.^24,28 Moreover, the 17β-protosteryl cation was also demonstrated by the catalysis of

Figure 1.10 Proposed mechanisms for C-ring expansion and D-ring

formation.

14

20,21-dehydrooxidosqualene.²⁹

The final step of lanosterol synthesis is the rearrangement of the protosteryl cation. After two hydride and two methyl groups shift (H-17α→20, H-13α→17α, CH3-14β→13β, CH3-8α→14α), this is followed by deprotonation on C-8 / C-9 to

Figure 1.11 The previous study proposed an incorrect C17α stereochemistry of protosteryl cation whereby the intermediate required a large side-chain rotation prior to rearrangement to account for the observed stereochemistry at C20.

17α-orientation

20R-product 120° rotation of C17-C20 bond

Figure 1.12 The evidence for the stereochemistry of protosteryl cation

intermediate at C17 preferring a β- rather than an α- orientation at C-17

position by incubation of substrate analogue with OSC.

15

generate the product lanosterol.

In 2004, Thoma and co-workers solved the X-ray structure of human OSC which is in complex with lanosterol.³⁰ Human OSC is a monomer that consists of two barrel domains that are connected by loops and three smaller β-structures, and the large active-site cavity is located in the center of the molecule between domains 1 and 2. It is a monotopic membrane protein that attached to the membrane from one side, and the membrane-inserted surface consists of a plateau with 25 Å in diameter and a channel that leads to the active-site cavity. The channel is considered to permit oxidosqualene, as a substrate, to enter the hydrophobic active site and have the separated role from that of the putative active-site cavity. Achievement of the substrate passage is constructed either by a change in the side chain of the residue such as Tyr237, Cys233 and Ile524 or by rearrangement of the strained loops from 516 to 524 and from 697 to 699 (Fig. 1.13). Because of the human OSC crystal structure, the relationship between the functional residues and cyclization mechanism could be understood in depth.

Figure 1.13 Human OSC structure. (a) The ribbon diagram of human OSC structure. 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

is shown relative to one leaflet of the membrane, and Ro48-8071 binds in

the central active-site cavity.

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1.3.4 Cycloartenol synthase (CAS)

Cycloartenol synthase (EC 5.4.99.8) is the sterol precursor in higher plants and its skeleton is similar to lanosterol. The reaction of converting oxidosqualene into cycloartenol is very similar to that catalyzed by lanosterol synthase. Moreover, cycloartenol synthase has 759 amino acids and the molecular mass is approximately 86 kDa. Although there are 400 amino acids that are different, shown in the results of sequence alignment between CAS and OSC, all steps in cycloartenol and lanosterol synthase are identical with the exception of the final deprotonation reaction.

Cycloartenol synthase forms the cyclopropyl ring and abstracts a proton from C-19, whereas lanosterol synthase removes a different proton and forms lanosterol (Fig.

1.14).

Phytosterols, such as campesterol and sitosterol, are biosynthesized from cycloartenol. Phytosterols, also called as plant sterols, are a group of naturally occurring steroid alcohols synthesized by plants. In addition to cholesterol-lowering effects, phytosterols have been suggested to possess anti-inflammatory, antibacterial, antifungal, antiulcerative, and antitumor activities.³¹

Cycloartenol synthase was first cloned from Arabidopsis thaliana (AthCAS1), and

(3S)-2,3-Oxidosqualene

Figure 1.14 The difference between cyclization mechanisms of lanosterol

synthase and cycloartenol synthase.

17

expressed and characterized in the yeast lanosterol synthase system in 1993.³² Furthermore, because the cyclopropyl ring formation in the cycloartenol biosynthesis is thermodynamically unfavorable relative to that of the lanosterol formation, some of amino acid differences are probably specifically required for inducing cyclopropyl ring formation and excluding the formation of more energetically favored products by the cycloartenol synthase. The site-direct mutagenesis study of cycloartenol synthase (AthCAS1) showed some important residues such as Tyr410, His477, and Ile481. ³³ These residues are highly-conserved within cycloartenol synthase in many plant species, but they maintain Thr, Cys or Gln, and Val in the corresponding positions of ERG7 from animals and fungi (Fig. 1.15). Speculation on this phenomenon, these residues may promote the formation of cyclopropyl rings within AthCAS1; in other words, mutations at these positions may permit the lanosterol formation (Table 1.1).

Ile481 is conserved in all cycloartenol synthases, whereas Val is present in lanosterol synthase at this position. The γ-methyl of Ile481 might promote cycloartenol formation by preventing the rotation of the intermediate cation through steric interactions with C-2 and the two axial methyl groups at the A-ring. Removing the γ-methyl group with an Ile481Val substitution resulted in 25% lanosterol production in addition to production of cycloartenol and parkeol. In addition, Ile481 may also be involved in assisting in proper substrate folding, as well as for the cyclization reaction.

Mutation of Ile481 to smaller residues such as Ala and Gly has led to achilleol A and camelliol C production (Fig. 1.16).³⁴

Figure 1.15 Conservation pattern between CAS1 and ERG7

AthCAS1 QGYNG 412 TADHGWPISDCT 485

DdiCAS1 QGYNG 365 TVDHGWPISDCT 437 SceERG7 MGTNG 386 TKTQGYTVADCT 458 SpoERG7 RGTNG 381 NITQGYTVSDTT 453 HsaERG7 QGTNG 383 TLDCGWIVSDCT 457 RnoERG7 QGTNG 384 TLDCGWIVADCT 458

18

Figure 1.16 Products formed by cycloartenol synthase mutants.

AthCAS1

^mut Cycloartenol Lanosterol Parkeol 9β-lanosta-

7,24-dien-3β-ol Achilleol A Camelliol C

Wild type 99 1

I481V/H477N/Y410T 78 22

I481V/H477Q/Y410T 78 22

I481V/H477N 99 1

Table 1.1 Product profiles of AthCAS Ile481, Tyr410 and His477 mutants.

Lanosterol

19

Tyr410 and His257 participate in an H-bonding network positioned near the C-19 methyl group for the deprotonation reaction.³³ Tyr410 is present in all cycloartenol synthases, but animal and fungal oxidosqualene-lanosterol synthase maintain Thr at the corresponding position. The AthCAS1^Tyr410Thr mutant forms 65% lanosterol, along with 9β-lanosta-7,24-dien-3β-ol and parkeol. Removing the aromatic ring of Tyr410 decreases the steric bulk above the intermediate cation. Because the hydroxyl group in Thr is closer to its α-carbon than that in Tyr, the polar groups of Tyr410Thr, Tyr532, and His257 were repositioned in the Tyr410Thr mutant. This combination of steric and electronic changes abolishes the cycloartenol synthesis and allows deprotonation of C-8/C-9 lanosterol cation to form lanosterol, parkeol, and 9β-lanosta-7,24-dien-3β-ol.³⁵

His477 is not in the active site, but is a second-sphere residue that affects the product profile through the interactions with the side chain of Tyr410.³³ His477 is strictly conserved in the known cycloartenol synthase, whereas lanosterol synthases maintain either Gln or Cys. The AthCAS1^His477Gln mutant has the polar functionality moved toward C-11, consequently resulting in more parkeol production than lanosterol. The AthCAS1^His477Asn mutant formed lanosterol by positioning the basic group near the C-9/C-8, but also produced parkeol due to a close enough distance to C-11.³⁶

The double mutant of CAS1I481V/ Y410T

formed lanosterol more accurately than either single mutant alone. However the triple mutant (His477Asn/Gln, Ile481Val, and Tyr410Thr) did not promote the lanosterol synthesis because the hydroxyl group of Thr is too distant to interact with the amide group of Asn or Gln residues. The His477Asn Ile481Val double mutant is the most accurate example for the enzyme mutation to generate lanosterol.³⁶

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1.3.5 β-amyrin synthase (βAS)

β-amyrin synthase originates from several plant species, such as Pisum

sativum and Panax ginseng, and it is also a member of phytosterols biosynthesis

pathway. The biosynthesis mechanism of β-amyrin is much different from that of lanosterol and cycloartenol. In the beginning, oxidosqualene is pre-folded into a chair-chair-chair conformation, and then forms the dammarenyl cation intermediate.

Next, the D-ring expansion and E-ring cyclization occur, producing the 6-6-6-6-5 pentacyclic lupenyl cation. The lupenyl cation then converts to lupeol by deprotonation by lupeol synthase. But for β-amyrin synthase, the E-ring expands to continue the rearrangement cascade. Through the last deprotonation step, the β-amyrin is generated.

Figure 1.17 Proposed mechanism of 2,3-oxidosqualene converted into

β-amyrin and lupeol.

21

Since the biogenetic isoprene rule proposed by Eschenmoser, Ruzicka, Arigoni, and Jeger in 1955,²⁷ the cyclization of (3S)-2,3-oxidosqualene into β-amyrin has fascinated organic chemists for over a half century. To investigate the catalytic motifs within cyclases that form the dammarenyl cation, the Ebizuba group first generated chimeras of the A. thaliana lupeol synthase and the Panax ginseng β-amyrin synthase.³⁷ They determined the function of the portions of cyclase by using a domain swapping strategy. It was observed that only relatively small portions of the protein can control the generation of lupeol or β-amyrin. One chimera in which only one fourth of the protein was β-amyrin sequence made four times as much β-amyrin as it did lupeol, and a mixed PCR method further confirmed the important region of chimeras. From an alignment analysis, it showed that the Trp259 within

Panax ginseng β-amyrin synthase (PNY) and the Leu256 within Olea europa lupeol

synthase (OEW) might control the product specificity.³⁸ Therefore, the authors constructed the PNY^Trp259Leu and OEW^Leu256Trp mutants and conducted the product analysis. Lupeol was twice as abundant as β-amyrin in the product profile of the PNY^Trp259Leu mutant, whereas β-amyrin was the major product for the OEW^Leu256Trp mutant. These results indicated that this position plays a critical role in directing either β-amyrin or lupeol formation. Furthermore, the authors created the PNY^Tyr261His mutant and the experimental results showed that Tyr261 stabilizes one of the cationic intermediates formed after the dammarenyl cation.^22,37-38 In addition, the experimental results for Pisum sativum β-amyrin synthase (PSY) showed that the expansion of the D-ring could take place in the absence of the terminal double bond, so that the formation of anti-Markovnikov six-membered D-ring is independent of the terminal π-electrons. Thereby, the aromatic residues within the putative active site might play a crucial role for the ring expansion reaction.²¹

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1.3.6 The amino acid sequence alignment

The amino acid sequence alignment of the enzyme cyclases could provide much information and led us understand these cyclases in depth. In evolutionary history, some highly-conserved residues indicated that they may play some specific function and it is necessary for the organisms. Therefore, it would be retained in various species. However, some other residues were not conserved or even deleted through the variation of evolution in many species. The information of these differences helped us to figure out the cyclized mechanism of enzyme cyclases.

Moreover, their similar structures, stereoselectivity, and the catalytic mechanism were also investigated.³⁹ The relationship between the function and structure thus were deeply realized.

In order to understand the functional residues within the putative active site of triterpene cyclases, the program of Clustal W had been used to produce multiple sequence alignment of the following enzymes: H. sapiens ERG7: P48449, S.

cerevisiae ERG7: P38604, A. thaliana CAS1: NP_178722, P. sativum PSY:

BAA97558, A. acidocaldarius SHC: BAA25185. All of them were obtained from the Protein Data Bank (PDB) in NCBI, and the result of sequence alignment is shown below.

H.sapiens ERG7 MTEGTCLRRRGGPYKTEPATDLG--RWRLN-CERGRQTWTYLQDER---AGREQT 49 S.cerevisiae ERG7 MTEFYSDTIG---LPKTDPR--LWRLRTDELGRESWEYLTPQQ---AANDPP 44 A.thaliana CAS1 MWKLKIAEGGS-PWLRTTNNHVGRQFWEFDPNLGTPEDLAAVEEARKSFSDNRFVQKHSA 59 P.sativum PSY MWRLKIAEGGNDPYLFSTNNFVGRQTWEYDPEAGSEEERAQVEEARRNFYNNRFEVKPCG 60 A.acidocaldarius SHC ---

H.sapiens ERG7 GLEAYALGLDTKNYFKDLPKAH---TAFEGALN----GMTFYVGLQAED-GHWTGDY 98 S.cerevisiae ERG7 STFTQWLLQDPK-FPQPHPERNKHSPDFSAFDACHN----GASFFKLLQEPDSGIFPCQY 99 A.thaliana CAS1 DLLMRLQFSRENLISPVLPQVKIEDTDDVTEEMVETTLKRGLDFYSTIQAHD-GHWPGDY 118 P.sativum PSY DLLWRFQVLRENNFKQTIGGVKIEDEEEITYEKTTTTLRRGTHHLATLQTSD-GHWPAQI 119 A.acidocaldarius SHC ---MAEQLVEAPAYARTLDRAV---EYLLSCQKDE-GYWWGPL 36

23

H.sapiens ERG7 GGPLFLLPGLLITCHVAR---IPLPAGYREEIVRYLRSVQLP-DGGWGLHIEDKSTVFGT 154 S.cerevisiae ERG7 KGPMFMTIGYVAVNYIAG---IEIPEHERIELIRYIVNTAHPVDGGWGLHSVDKSTVFGT 156 A.thaliana CAS1 GGPMFLLPGLIITLSITGALNTVLSEQHKQEMRRYLYNHQNE-DGGWGLHIEGPSTMFGS 177 P.sativum PSY AGPLFFMPPLVFCVYITGHLDSVFPPEHRKEILRYIYCHQNE-DGGWGLHIEGHSTMFCT 178 A.acidocaldarius SHC LSNVTMEAEYVLLCHILDR----VDRDRMEKIRRYLLHEQRE-DGTWALYPGGPPDLDTT 91

H.sapiens ERG7 ALNYVSLRILGVGPDDP---DLVRARNILHKKGGAVAIPSWGKFWLAVLNVYSWEGLNTL 211 S.cerevisiae ERG7 VLNYVILRLLGLPKDHP---VCAKARSTLLRLGGAIGSPHWGKIWLSALNLYKWEGVNPA 213 A.thaliana CAS1 VLNYVTLRLLGEGPNDG-DGDMEKGRDWILNHGGATNITSWGKMWLSVLGAFEWSGNNPL 236 P.sativum PSY ALNYICMRILGEGPDGGEDNACVRARNWIRQHGGVTHIPSWGKTWLSILGVFDWLGSNPM 238 A.acidocaldarius SHC IEAYVALKYIGMSRDEE---PMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMV 148

H.sapiens ERG7 FPEMWLFPDWAPAHPSTLWCHCRQVYLPMSYCYAVRLSAAEDPLVQSLRQELYVEDFASI 271 S.cerevisiae ERG7 PPETWLLPYSLPMHPGRWWVHTRGVYIPVSYLSLVKFSCPMTPLLEELRNEIYTKPFDKI 273 A.thaliana CAS1 PPEIWLLPYFLPIHPGRMWCHCRMVYLPMSYLYGKRFVGPITSTVLSLRKELFTVPYHEV 296 P.sativum PSY PPEFWILPSFLPMHPAKMWCYCRLVYMPMSYLYGKRFVGPITPLILQLREELHTEPYEKI 298 A.acidocaldarius SHC PPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVP--ELYETDVPPR 206

H.sapiens ERG7 DWLAQRNNVAPDELYTPHSWLLRVVYALLNLYEHHHS---AHLRQRAVQKLYEHIVA 325 S.cerevisiae ERG7 NFSKNRNTVCGVDLYYPHSTTLNIANSLVVFYEKYLRN---RFIYSLSKKKVYDLIKT 328 A.thaliana CAS1 NWNEARNLCAKEDLYYPHPLVQDILWASLHKIVEPVLMRWPG-ANLREKAIRTAIEHIHY 355 P.sativum PSY NWTKTRHLCAKEDIYYPHPLIQDLIWDSLYIFTEPLLTRWPFNKLVRKRALEVTMKHIHY 358 A.acidocaldarius SHC RRGAKGG---GGWIFDALDRALHGYQKLSVHP---FRRAAEIRALDWLLE 250

H.sapiens ERG7 DDRFTKSISIGPISKTINMLVRWYVDGPASTAFQEHVSRIPDYLWMGLDGMKMQGTNGSQ 385 S.cerevisiae ERG7 ELQNTDSLCIAPVNQAFCALVTLIEEGVDSEAFQRLQYRFKDALFHGPQGMTIMGTNGVQ 388 A.thaliana CAS1 EDENTRYICIGPVNKVLNMLCCWVED-PNSEAFKLHLPRIHDFLWLAEDGMKMQGYNGSQ 414 P.sativum PSY EDENSRYLTIGCVEKVLCMLACWVED-PNGDAFKKHIARVPDYLWISEDGMTMQSF-GSQ 416 A.acidocaldarius SHC RQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISP 310

H.sapiens ERG7 IWDTAFAIQALLEAGGHHRPEFSSCLQKAHEFLRLSQVP-DNPPDYQKYYRQMRKGGFSF 444 S.cerevisiae ERG7 TWDCAFAIQYFFVAGLAERPEFYNTIVSAYKFLCHAQF---DTECVPGSYRDKRKGAWGF 445 A.thaliana CAS1 LWDTGFAIQAILATNLVE--EYGPVLEKAHSFVKNSQVLEDCPGDLNYWYRHISKGAWPF 472 P.sativum PSY EWDAGFAVQALLATNLIE--EIKPALAKGHDFIKKSQVTENPSGDFKSMHRHISKGSWTF 474 A.acidocaldarius SHC VWDTGLAVLALRAAGLPAD---HDRLVKAGEWLLDRQIT--VPGDWAVKRPNLKPGGFAF 365

24

H.sapiens ERG7 STLDCGWIVSDCTAEALKAVLLLQEK--CPHVTEHIPRERLCDAVAVLLNMRNPD----G 498 S.cerevisiae ERG7 STKTQGYTVADCTAEAIKAIIMVKNSPVFSEVHHMISSERLFEGIDVLLNLQNIGSFEYG 505 A.thaliana CAS1 STADHGWPISDCTAEGLKAALLLSKVP-KAIVGEPIDAKRLYEAVNVIISLQNAD----G 527 P.sativum PSY SDQDHGWQVSDCTAEGLKCCLLLSLLP-PEIVGEKMEPERLFDSVNLLLSLQSKK----G 529 A.acidocaldarius SHC QFDNVYYPDVDDTAVVVWALNTLRLPD---ERRRRDAMTKGFRWIVGMQSSN----G 415

H.sapiens ERG7 GFATYETKRGGHLLELLNPSEVFGDIMIDYTYVECTSAVMQALKYFHKRFPEHRAAEIRE 558 S.cerevisiae ERG7 SFATYEKIKAPLAMETLNPAEVFGNIMVEYPYVECTDSSVLGLTYFHKYF-DYRKEEIRT 564 A.thaliana CAS1 GLATYELTRSYPWLELINPAETFGDIVIDYPYVECTSAAIQALISFRKLYPGHRKKEVDE 587 P.sativum PSY GLAAWEPAGAQEWLELLNPTEFFADIVVEHEYVECTGSAIQALVLFKKLYPGHRKKEIEN 589 A.acidocaldarius SHC GWGAYDVDNTSDLPNHIPFCDFG--EVTDPPSEDVTAHVLECFG---SFGYDDAWK 466

H.sapiens ERG7 TLTQGLEFCRRQQRADGSWEGSWGVCFTYGTWFGLEAFACMGQTYRDGTACAEVSRACDF 618 S.cerevisiae ERG7 RIRIAIEFIKKSQLPDGSWYGSWGICFTYAGMFALEALHTVGETYEN---SSTVRKGCDF 621 A.thaliana CAS1 CIEKAVKFIESIQAADGSWYGSWAVCFTYGTWFGVKGLVAVGKTLKN---SPHVAKACEF 644 P.sativum PSY FIFNAVRFLEDTQTEDGSWYGNWGVCFTYGSWFALGGLAAAGKTYTN---CAAIRKGVKF 646 A.acidocaldarius SHC VIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREP----YIQKALDW 522

H.sapiens ERG7 LLSRQMADGGWGEDFESCEERRYLQSA--QSQIHNTCWAMMGLMAVRHPDIE--AQERGV 674 S.cerevisiae ERG7 LVSKQMKDGGWGESMKSSELHSYVDSE--KSLVVQTAWALIALLFAEYPNKE--VIDRGI 677 A.thaliana CAS1 LLSKQQPSGGWGESYLSCQDKVYSNLDGNRSHVVNTAWAMLALIGAGQAEVDRKPLHRAA 704 P.sativum PSY LLTTQREDGGWGESYLSSPKKIYVPLEGNRSNVVHTAWALMGLIHAGQSERDPTPLHRAA 706 A.acidocaldarius SHC VEQHQNPDGGWGEDCRSYEDPAYAGKG--ASTPSQTAWALMALIAGGRAESE--AARRGV 578

H.sapiens ERG7 RCLLEKQLPNGDWPQENIAG-VFNKSCAISYTSYRNIFPIWALGRFSQLYPERALAGHP 732 S.cerevisiae ERG7 DLLKNRQEESGEWKFESVEG-VFNHSCAIEYPSYRFLFPIKALGMYSRAYETHTL---- 731 A.thaliana CAS1 RYLINAQMENGDFPQQEIMG-VFNRNCMITYAAYRNIFPIWALGEYRCQVLLQQGE--- 759 P.sativum PSY KLLINSQLEQGDWPQQEITG-VFMKNCMLHYPMYRDIYPLWALAEYRRRVPLP--- 758 A.acidocaldarius SHC QYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR--- 631

Figure 1.18 Amino acids alignment. The red words are the CAS1

^mut

and the green one is ERG7

^C703

mutation.

25

1.4 Research motive

The oxidosqualene cyclases family and their sterol products have fascinated scientists for more than half of a century. Not only biochemists, but also organic chemists and physical chemists, even pharmaceutical scientists and doctors are conducting research topic on this family of molecules. The goals of these studies are to understand the enzyme mechanisms, to make artificial enzymes to synthesize specific products, to find inhibitors to against bacteria or fungi, and to produce drugs

The oxidosqualene cyclases family and their sterol products have fascinated scientists for more than half of a century. Not only biochemists, but also organic chemists and physical chemists, even pharmaceutical scientists and doctors are conducting research topic on this family of molecules. The goals of these studies are to understand the enzyme mechanisms, to make artificial enzymes to synthesize specific products, to find inhibitors to against bacteria or fungi, and to produce drugs

在文檔中 利用定點突變進行氧化鯊烯環化酵素家族假設活性區中胺基酸之特性探討 (頁 22-0)