3.2 Functional analysis of ERG7 W443 within S. cerevisiae
3.2.4 Analysis of the ERG7 W443X in the OSC homology modeling …
The multiple sequence alignment analysis showed that the Trp443 was highly conserved in most cyclase, the Trp443 of S. cerevisiae ERG7 corresponds to F363 in A.
acidocaldarius SHC and to W470 in A.thaliana CAS. These three residues are all aromatic
amino acids; however their functional role analysis during catalytic cyclization mechanism has not been suggested and reported before.
According to the previous studies, the achilleol A and camelliol C were also produced from the other cyclase-inactive mutants, Lys448Ala which were previously identified from the region upstream of the putate active site in our laboratory by ergosterol complement experiment. Lys448 are located at the flexible loop region opposite to the position of the essential Asp456 and displayed interactions to hold the correct conformation in dimeric association with two amino acids, Phe426 and Asn332. Replacing Lys448 with Ala was supposed to disrupt the electrostatic interaction between subunits or held the cyclization/rearrangement cascade at the intermediate stage, thus forming only the initially cyclized A-ring.[62-63]
In the previous homology model studies, the Trp443 was supposed to be positioned spatially opposite to the Asp456, below the molecular plain and close to the high-energy C-10 (lanosterol numbering) cationic intermediate. The Trp443 was suggested to be at the nearest neighbor to the active site residues and thereby stabilize the high-energy C-10 cation intermediate during the concerted process of epoxide opening and A-ring formation.
Substitution of Trp with Ala might disrupt the steric or cation- electronic effect between substrate and enzyme; elongation of the cyclization cascade would thus be inhibited and the reaction be held at monocyclic triterpenes.[62-63] However, in my homology modeling analysis, the Trp443 is positioned spatially above to the Asp456 and the molecular plain whereas it seems to be far from substrate (10.28Å between oxygen of Trp and C-10 of lanosterol; 10.1Å between oxygen of Trp and C-2 of lanosterol). (Fig. 3.15)
Figure 3.15 Local views of the homology modeled Asp456, Trp443, Lay448, and Phe445 positions in S. cerevisiae ERG7 structure based on the X-ray structure of lanosterol-complexed human OSC and determined by using the Insight II Homology program.
Obviously, Trp443 is not located in the putative π–electron pocket; however the site-saturated mutagenesis results showed that only W443Val, W443Leu (aliphatic residues), W443Cys, W443Met (sulfur-containing residues), W443Phe (aromatic residue) and His (basic residue) were able to complement to the OSC deficient strain and yield lanosterol. This observation revealed that W443 position is indispensable; changes of the side chain at W443 position may influence the interactive distances for the proper substrate binding and subsequent catalysis. On the other hand, two inactive mutants, W443A (aliphatic residue) and W443K (basic residue) produced achilleol A as a major product and camelliol C as a minor product. The formation of the truncated monocyclic intermediated suggested that Trp residue may also play an crucial role both in influencing the substrate
prefolding and stabilizing the epoxide protonation and inducing A-ring formation via the generation of the C-10 cation.
Moreover, the exact reason for the higher accumulation of achilleol A over that of camelliol C, and the production of achilleol A whenever camelliol C is produced, remain unclear. Furthermore, whether the Trp443 interact with the substrate directly or via the other residues in the active site pocket, needs more mutagenesis at neighboring residues and homology modeling of the W443X mutants, in order to clarify the functional roles of Trp443.
Chapter4 Conclusions
Site-directed mutagenesis is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule, and site-saturated mutagenesis means the substitution of specific sites with other 19 proteinogenic amino acids. This technique was applied to obtain a detailed understanding of structure-function relationships for the putative active sites in the enzyme.
In our studies, site-saturated mutagenesis coupled with product isolation and characterization of the mutations at Tyr99 and Trp443 position of OSC ERG7 within S.
cerevisias revealed their catalytic function in affecting the cyclization/rearrangement
mechanism. Both of these two residues were suggested play crucial roles in enzyme catalytic cyclization/rearrangement. Herein we summarize several important conclusions of our studies:
4.1 The functional analysis of TKW14C2[pERG7
Tyr99X]
(1) The TKW14C2[pERG7Tyr99X] expressed ERG7Tyr99X as its sole oxidosqualene cyclase.
The genetic selection results showed that several Tyr99X mutants could complement to ergosterol-deficient growth except the deletion of Tyr99 as well as the mutation of Y99N, Y99H in ERG7.
(2) Several mutants including Y99A, Y99G, Y99I, Y99D, Y99S, Y99T, Y99F, and Y99P produced two novel products with a molecular mass of m/z = 426 except lanosterol, which are both truncated tricyclic triterpenoid products (13αH)-isomalabarica-14Z, 17E, 21-trien-3β-ol and (13αH)-isomalabarica-14E, 17E, 21-trien-3β-ol, identified by 1H and
13C NMR for the first time. The product profile of ERG7Y99X demonstrates the truncation of the cyclization/rearrangement cascade at chair-boat 6-6-5 tricyclic
C-15 position with different stereochemical preferences.
(3) The functional role of ERG7Y99 is suggested to affect both chair-boat 6-6-5 tricyclic Markovnikov cation stabilization and the stereochemistry of the protons at the C-15 position for subsequent deprotonation, but not to enforce the boat conformation for lanosterol B-ring formation.
(4) In homology modeling analysis, the phenolic oxygen of Tyr99 residue is at a distance of approximately 4.4Å from the C-14 cation, and its location is differently in space from that to His234 and Phe445 to the common C-14 cation which affects the orientation or electrostatic interaction between the enzyme and its cationic intermediate, and results in the abstraction of a proton form a different position or orientation.
Therefore, changes at Tyr99 or a deletion of this position may strongly impact the structure and lead to an adjustment of the active site and result in obstruction of substrate binding and catalysis.
(5) The product energy profile from quantum mechanical calculations suggested that the energetics of stereochemical control during the tricyclic Markovnikov cation deprotonation step could be affected by the inclusion of these enzymatic effects. It may be the reason why the (13αH)-isomalabarica-14Z, 17E, 21-trien-3β-ol was produced as a major product in Y99Gly, Y99Ala, Y99Ser, Y99Thr and Y99Pro mutants.
4.2 The functional analysis of TKW14C2[pERG7
Trp443X]
(1) In the previous alanine-scanning mutagenesis and plasmid shuffle selection of the
441GAWGFSTKTQGYT453 within this of S. cerevisiae ERG7, Trp443Ala was one of the inactive mutants. The following ergosterol complementation experiment uncovered that ERG7Trp443Ala produced two monocyclic triterpenen products concomitantly, achilleol A and camelliol C.
(2) The genetic selection demonstrated that only six mutants including Trp443Val, Trp443Leu, Trp443His, Trp443Cys, Trp443Met, and Trp443Phe which allowed for ergosterol-independent growth and yielded lanosterol as an only product with molecular mass of m/z=426. Whereas one of inactive mutant, Trp443Lys, revealed two monocyclic triterpenoid products with a molecule mass of m/z = 426: achilleol A and camelliol C, as well as the observation in the ERG7W443A mutant.
(3) The formation of achilleol A and camelliol C were identified as evidence for premature truncation of C-10 cationic intermediates following the proton abstraction from Me-25 or C-1 position. This finding suggested that Trp residue may also play a crucial role both in influencing the substrate prefolding and stabilizing the epoxide protonation and A-ring formation to generate C-10 cation. However, the exact reason for the higher accumulation of achilleol A over that of camelliol C, and the production of achilleol A whenever camelliol C is produced, remain unclear.
(4) Although Trp443 is not located in the putative π–electron pocket and positioned spatially far from A-ring of lanosterol, it might provide an interaction with the neighboring residues to stabilize the carbocationic intermediates produced during protonation of epoxide and subsequent A-ring formation.
Chapter 5 Future Works
For the Tyr99 functional analysis, our results showed that how the structure-function relationships of the OSC via the expression of ERG7Y99X site-saturated mutants in S.
cerevisiae. However, our substantiation of Tyr99 functional role contradicted the
supposition, which Tyr98 of human OSC is spatially positioned to enforce the energetically unfavorable boat conformation of OS for lanosterol B-ring formation via pushing the methyl group at C-8 (lanosterol numbering) below the molecular plane. The expression of site-directed mutants of Tyr98XhOSC will be carried out to identify the function of this conserved residue.
Furthermore, the HEM1 ERG7 ERG1 triple knockout mutant which is the yeast strain with the deletion of both oxidosqualene cyclase (ERG7) and squalene epoxidase (ERG1). This triple knockout strain will be developed for the in vitro analysis of the mutated oxidosqualene cyclase via the addition of the substrates. This oxidosqualene free strain will prevent the interference due to the downstream enzymes and consequently ensure the more detailed understanding for the catalytic function of the putative active-sites.
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Appendix 1
Primers used in this thesis
MutagenesisWSL-OSC-Y99SMC-1 5’-CCg TgT CAA (A/T)(T/g)(g/T) AAA ggg CCC ATg TTC ATg-3’
WSL-OSC-Y99SMC-2 5'-CAT gAA CAT ggg CCC TTT (C/A)(C/A)(T/A) TTg ACA Cgg-3'
WSL-OSC-Y99EVQ-1 5’-CCg TgT CAA (C/g)(A/T)(g) AAA ggg CCC ATg TTC ATg-3’
WSL-OSC-Y99EVQ-2 5'-CAT gAA CAT ggg CCC TTT (C)(T/A)(C/g) TTg ACA Cgg-3'
WSL-OSC-delY99-1 5’-CCg TgT CAA AAA ggg CCC ATg TTC ATg-3’
WSL-OSC-delY99-2 5'-CAT gAA CAT ggg CCC TTT TTg ACA Cgg-3'
FHC-W443A 5'-gA AAg ggg gCT ATg ggC TTC TCA ACA AAA ACC CAA ggC TAT ACA gTg g -3'
WSL-OSC-W443M-1 5’-gA AAg ggg gCT ATg ggC TTC TCA ACA AAA ACC CAA ggC TAT ACA gTg g-3’
WSL-OSC-W443M-2 5'-C CAC TgT ATA gCC TTg ggT TTT TgT TgA gAA gCC CAT AgC CCC CTT T C-3'
HJW-W443IMK 5'-TTT AAT TgC TTC TgC AgT gCA ATC TgC CAC TgT ATA gCC TTg ggT TTT TgT TgA gAA gCC (C/T)(A/T)T AgC CCC CTT TC-3'
HJW-W443VDEG 5'-TTT AAT TgC TTC TgC AgT gCA ATC TgC CAC TgT ATA gCC TTg ggT TTT TgT TgA gAA gCC (g/T)(A/C/T)C AgC CCC CTT TC-3'
HJW-W443FC 5'-TTT AAT TgC TTC TgC AgT gCA ATC TgC CAC TgT ATA gCC TTg ggT TTT TgT TgA gAA gCC g(A/C)A AgC CCC CTT TC-3'
HJW-W443Q 5'-TTT AAT TgC TTC TgC AgT gCA ATC TgC CAC TgT ATA gCC TTg ggT TTT TgT TgA gAA gCC CTg AgC CCC CTT TC-3'
Sequencing
HJW-L158-A2 5'-gAT TAC ATA gTT CgC TAC ggT Acc AAA CAC-3'
YTL-OSCW390X-1 5'-CCA TTA Tgg gTA CCA Atg gTg TgC AAA CCN NNg ATT gTg Cg-3'
Appendix 2
DNA electrophoresis of site-directed mutagenesis
(a)Appendix 2. DNA agarose gel electrophoresis of site-directed mutated plasmid checked by restriction enzymes. Lane M, 10-100 bp. DNA marker. Lane WT, pRS314+ERG7. (a) Lane 1-10 pRS314+ERG7Y99X. (b) Lane 1-6 pRS314+ERG7W443X.
Appendix 3
1
H NMR of (13αH)-isomalabarica-14Z,17E,21-trien-3β-ol
13
C NMR of (13αH)-isomalabarica-14Z,17E,21-trien-3β-ol
DEPT of (13αH)-isomalabarica-14Z,17E,21-trien-3β-ol
DEPT-135
DEPT-90
13C spectrum
HMQC of (13αH)-isomalabarica-14Z,17E,21-trien-3β-ol
HMBC of (13αH)-isomalabarica-14Z,17E,21-trien-3β-ol
1
H-
1H COSY of (13αH)-isomalabarica-14Z,17E,21-trien-3β-ol
Appendix 4
1
H NMR of (13αH)-isomalabarica-14E,17E,21-trien-3β-ol
13
C NMR of (13αH)-isomalabarica-14E,17E,21-trien-3β-ol
DEPT of (13αH)-isomalabarica-14E,17E,21-trien-3β-ol
DEPT-135
DEPT-90
13C spectrum
HMQC of (13αH)-isomalabarica-14E,17E,21-trien-3β-ol
HMBC of (13αH)-isomalabarica-14E,17E,21-trien-3β-ol
1