* H W-M 1,3-alkyl shift * * *

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2 FPP squalene synthase allylic cation 1' PP 2' 3' FPP electrophilic addition results tertiary cation PP loss of proton with formation of cyclopropane ring PP presqualene PP loss of diphosphate results in primary cation W-M 1,3-alkyl shift 1,3-alkyl shift generates new cyclopropane ring and more favorable tertiary cation bond cleavage produces alkene and favorable allylic cation (NADP) cation quenched by attack of anhydride Figure by MIT CW. squalene

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4 squalene 2 NADP cyclizations squalene oxide protonation of epoxide electrophilic addition electrophilic addition allows ring opening to gives tertiary cation + gives tertiary cation + tertiary cation six-membered ring six-membered ring squalene oxide electrophilic addition gives tertiary cation + five-membered ring protosteryl cation electrophilic addition gives tertiary cation W-M rearrangement; ring expansion despite tertiary --> secondary cation Figure by MIT CW

5 chair - boat - chair - boat squalene oxide sequence of W-M 1,2-hydride and 1,2-methyl shifts protosteryl cation sequence of W-M 1,2-hydride and 1,2-methyl shifts cyclopropane ring protosteryl cation loss of -9 creates formation and double bond loss of proton from C-10 methyl cycloartenol lanosterol Figure by MIT CW

6 Aromatase-catalyzed conversion of androstenedione to estrone C 3 R S NADP NADP NADP C + A Three possible mechanisms for the last step in the aromatase-catalyzed oxygenation of androstenedione. 1 Fe Fe Fe +3 Fe +3 Fe A +4 Fe +4 Fe 2 A + +4 Fe C Fe 3+ + C +4 Fe +4 Fe C 3 +4 Fe +4 Fe C Fe 3+ A + + C +4 Fe Figure by MIT CW.

7 PLANT C-24 alkylation loss of C -4α opening of methyl cyclopropane loss of C-14α methyl and c y c l o a r t en o l 24-met h ylen ec y c lo ar t a n o l c y c lo eu c a len o l o b t u s ifo l i o l allylic isomerization (migration of Δ 8 to Δ 7 ) 24-ethyl ste r o ls loss of C-4 methyl further sidechain alkylation av e n a s t e r o l c itrostad i e n ol gr a mi s t e r o l ( 24-et hy liden elo ph e n o l) ( 24-m e th y l en elo p h e n o l) loss of C-4 methyl epis ter o l 24-methyl sterols YEAST C-24 alkylation C-14 demethylation demethylations as C-4 side chain and ring B lanosterol eburicol 4,4-dimethylfecosterol fecosterol modifications (24-methylenedihydrolanosterol) Figure by MIT CW. ergosterol

8 cyclization is initiated by protonation of a double bond resulting in tertiary cation + squalene rings A and B are formed as 5- membered rings via Markovnikov addition; they then expand to 6- membered rings via W-M rearrangements A B 2 2 Figure by MIT CW hopan-22-ol tetrahymanol

9 GGPP allylic cation PP PP GGPP electrophilic addition resulting in tertiary cation loss of proton with formation of cyclopropane ring loss of diphosphate PP results in primary cation prephytoene PP W-M 1,3-alkyl shift generates new cyclopropane ring and more favorable tertiary cation proton loss generates alkene bond cleavage produces alkene and favorable allylic cation Z Z-phytoene sequence of desaturation reactions; in plants and fungi, the central double bond is also isomerized Z --> E E lycopene Figure by MIT CW.

10 + protonation of double bond results in tertiary cation; then electrophilic addition 2 NADP a ε-ring c b b opening of pinacol-like rearrangement a γ-ring epoxide ring generates ketone c 2 2 NADP NADP + β-ring 2 NADP + allene generated from opening of epoxide ring and loss of proton (a) (b) Figure by MIT CW. β-carotene central cleavage generates two molecules of retinal central cleavage (a) oxidative chain shortening excentric cleavage (b) excentric cleavage can generate one molecule of retinal retinal

11 Abietadiene Synthase bifunctional diterpene cyclase from fir cannot separate functional domains cloned (a.a. sequence is) Figure removed due to copyright reasons. don't know structure "homology modeling' ep.-aristolochene synthase enzyme as a model (sequiterpene synthase)

12 Gibberrellin phytohormone bifunctional enzyme in fungi two enzymes in plant 2 individual enzymes igher plants protonation ionization PP CPS PP KS FCPS/KS FCPS/KS Phaeosphaeria sp. L487 yeast Figure by MIT CW. 1 bifunctional enzyme Figure by MIT CW.

13 Index of figures removed due to copyright reasons Jennewein, S., and R. Croteau. Figure 2 in Taxol: biosynthesis, molecular genetics, and biotechnological applications. Applied Microbiol Biotech 57 (2001): Jennewein, Stefan et al. Random sequencing of an induced Taxus cell cdna library for identification of clones involved in Taxol biosynthesis. PNAS 101 (2004): Brown, Geoffrey D. Scheme 8 in The biosynthesis of steroids and triterpenoids. Natural Product Reports 15 (1998): Abe, Ikuro, Michel Rohmer, and Glenn D. Prestwich. Scheme VI in Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes. Chemical Reviews 93 (1993): Rilling,.C., and Chayet, L.T. The 19-reaction conversion of lanosterol to cholesterol. Danielsson,., and Sjovall, J., eds. Sterols and Bile Acids. New York, NY: Elsevier, ISBN: oshino, Tsutomu, and Tsutomu Sato. Figure 3 in Squalene hopene cyclase: catalytic mechanism and substrate recognition. Chemical Communications (2002): Peters, Reuben J. Scheme 1, Figure 1, and Table 1 in Bifunctional Abietadiene Synthase: Mutual Structural Dependence of the Active Sites for Protonation-Initiated and Ionization-Initiated Cyclizations. Biochemistry 42 (2003): Williams, David C. et al. Figures 1, 3 and 6 in Intramolecular proton transfer in the cyclization of geranylgeranyl diphosphate to the taxadiene precursor of taxol catalyzed by recombinant taxadiene synthase. Chemistry & Biology 7 (2000):