A nematode sterol C4α-methyltransferase catalyzes a new methylation reaction responsible for sterol diversity

Primitive sterol evolution plays an important role in fossil record interpretation and offers potential therapeutic avenues for human disease resulting from nematode infections. Recognizing that C4-methyl stenol products [8(14)-lophenol] can be synthesized in bacteria while C4-methyl stanol products (dinosterol) can be synthesized in dinoflagellates and preserved as sterane biomarkers in ancient sedimentary rock is key to eukaryotic sterol evolution. In this regard, nematodes have been proposed to convert dietary cholesterol to 8(14)-lophenol by a secondary metabolism pathway that could involve sterol C4 methylation analogous to the C2 methylation of hopanoids (radicle-type mechanism) or C24 methylation of sterols (carbocation-type mechanism). Here, we characterized dichotomous cholesterol metabolic pathways in Caenorhabditis elegans that generate 3-oxo sterol intermediates in separate paths to lophanol (4-methyl stanol) and 8(14)-lophenol (4-methyl stenol). We uncovered alternate C3-sterol oxidation and Δ7 desaturation steps that regulate sterol flux from which branching metabolite networks arise, while lophanol/8(14)-lophenol formation is shown to be dependent on a sterol C4α-methyltransferse (4-SMT) that requires 3-oxo sterol substrates and catalyzes a newly discovered 3-keto-enol tautomerism mechanism linked to S-adenosyl-l-methionine-dependent methylation. Alignment-specific substrate-binding domains similarly conserved in 4-SMT and 24-SMT enzymes, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of methyl sterols. The combination of these results provides evolutionary leads to sterol diversity and points to cryptic C4-methyl steroidogenic pathways of targeted convergence that mediate lineage-specific adaptations.

for key to structures 2 TL, this laboratory, CM, commercial source. 3,4 Retention time of sterol relative to the retention of cholesterol in GC; M + is the molecular ion determined in mass spectroscopy equivalent to the molecular weight of compound.  57-69 (1986). GC RRTc's may differ here from those in Supplementary Table 1 due to slightly different GC columns and operating conditions used in the studies. Refer to the paper for details on methods and instrumentations. 2 RRTc determined by GC, R f determined by TLC, M+ determined by mass spectroscopy and absorption λmax determined by UV using a HPLC-UV system equipped to a diode array detector; E.A. is end absorption around 210 nm.   1987-1989 (1978). Compound numbers in SFig. 1 are not met to correlate with any other set of compound numbers in this paper.

Supplementary-sterol synthesis details.
All reactions (non-aqueous) were performed under an inert atmosphere of N 2 unless stated otherwise. Aluminum isopropoxide was purified via distillation as was toluene which was first dried with calcium sulfate and then distilled over molecular sieves. Cholesterol and 6ketocholestanol were purchased from Sigma Aldrich and were not purified any further. Each reaction was monitored by the use of thin layer chromatography (TLC) which was performed on Whatman silica gel aluminum backed plates (F-254). TLC was visualized with a dilute (2%) sulfuric acid stain and heat. Melting points were measured with a Thomas Hoover capillary melting point apparatus. All reagents were reagent grade unless otherwise specified.

6-Fluorocholesterol
To the established starting compound-6-ketocholestanol (220 mg, 0.546 mmol) was acetylated by adding 7 mL of pyridine and 2.5 mL of acetic anhydride at 0 o C. The reaction mixture was stirred at room temperature for 17 hours and the resulting reaction mixture was poured over an ice/water mixture. The desired organics were extracted with ether, then combined the organic layers were dried over magnesium sulfate, filtered and concentration in vacuo to yield crude 3acetoxycholest-6-one. 3-Acetoxycholest-6-one was then dissolved in dimethoxyethane (glyme) (7 mL) in a HDPE Teflon coated container and two drops of fuming sulfuric acid (70%) was added. Diethylaminosulfurtriflouride (DAST) (0.5 mL) was added to the reaction mixture and the reaction was heated at 50 o C for 21 days under an atmosphere of nitrogen. The reaction mixture was carefully poured into a 5% sodium bicarbonate solution with ice. The desired organics were extracted with ether, the combined organic layers were dried over magnesium sulfate, concentrated in vacuum to yield crude 3-acetoxy-6-fluorocholesterol. The crude material was purified by column chromatography on silica gel eluting with 6-25% ether: petroleum ether to yield pure 3-acetoxy-6-fluorocholesterol (mp 100 o C; lit. M.P. ~ 95 o -100 o C). 1 3-Acetoxy-6fluorocholesterol was immediately deacetylated by mixing with 10% potassium hydroxide in methanol (4mL). The reaction mixture was then extracted with ether (x3) and the organic layers were dried with magnesium sulfate and concentrated in vacuo to yield 6-fluorocholesterol as a colorless solid (52.4 mg, 24% yield over three steps  56.4, 56.4, 56.0, 49.8, 42.3, 39.5, 37.6, 36.1, 36.1, 35.7, 32.2, 32.0, 31.8, 28.1, 28.0, 24.2, 23.8, 22.8, 22.6, 21.1, 19.3, 19.3, 18.7, 11.8 Fresh cholest-4-ene-3-one To cholesterol (2.0 g, 5.17 mmol) was added toluene (60 mL), a solution of purified aluminum isopropoxide and dry toluene (0.33 g/mL) (37 mL) and cyclohexanone (25 mL). This reaction mixture was heated at 130 o C for 1 hour and 40 minutes. The reaction mixture was then cooled to room temperature and diluted with toluene. The mixture was then washed with sodium potassium tartate, dried over MgSO 4 and the organics were extracted with ether. The combined organic extracts were then concentrated in vacuo and the crude material was purified by column chromatography using silica gel as the stationary phase and an eluting gradient of 0-18% ether:petroleum ether to yield cholest-4-3-one (1.91 g 96%).

Cholest-4,7-diene-3-one
To 7-dehydrocholesterol (1.2 g, 3.12 mmol) was added toluene (25 mL), a solution of purified aluminum isopropoxide and dry toluene (0.33 g/mL) (22 mL) and cyclohexanone (60 mL). This reaction mixture was heated at 130 o C for 50 minutes. The reaction mixture was then cooled to room temperature and diluted with toluene. The mixture was then washed with sodium potassium tartate and was extracted with ether. The combined organic extracts were then concentrated in vacuo and purified by column chromatography using silic gel as the stationary phase and and an eluting gradient of 0-20% ether:petroleum ether to yield cholest-4,7-diene-3one (0.46 g, with trace cholesta-5,7-dien-3-one).

Fresh cholestanol
Cholesterol (0.580 g, 1.50 mmol) was dissolved in ethyl acetate (EtOAc)and a pinch of 10% Pd/C was added. The reaction mixture was evacuated under vacuum and replenished with an atmosphere of hydrogen gas (X3). The reaction mixture was allowed to stir overnight at room temperature. The reaction mixture was then diluted with EtOAc and filtered through a bed of celite. The filtrate was then evacuated in vacuo to yield crude material that was used for the next step.

Fresh cholestanone
Crude cholestanol (assume 100% from previous step), PCC (0.810 g, 3.76 mmol) and celite (0.4 g) were suspended in dichloromethane (DCM) and the reaction mixture was allowed to stir at room temperature for 3 h under an atmosphere of nitrogen. The resulting mixture was then filtered through a short bed of celite and the crude was dry loaded onto silica and purified by column chromatography using silica gel as the stationary phase and an eluting gradient of 9-20% ether:petroleum ether to yield cholestanone as a colorless solid (432 mg, 75% for two steps).

2α-Methyl-5α-cholestan-3-one, 2β-methyl-5α-cholestan-3-one and 4α-methyl-5α-cholestan-3one and 4β-methyl-5α-cholestan-3-one
Cholestanone (432 mg, 1.12 mmol) was dissolved in dry THF (8 mL) and the reaction mixture was cooled to -78 o C. LDA (1.0 M in THF) was added dropwise to the reaction mixture and the resulting reaction mixture was allowed to stir at -78 o C for 50 min under an atmosphere of nitrogen. HMPA (0.6 mL) was then added dropwise to the reaction mixture. A solution of methyl iodide (0.80 g, 5.6 mmol) in 3 mL dry THF was then added dropwise to the previously mentioned reaction mixture at -78 o C and the resulting reaction mixture was allowed to stir at -78 o C for 2.5 h under an atmosphere of nitrogen. To the reaction mixture was carefully added 10% citric acid and the reaction mixture was allowed to stir 20 minutes, while warming up to room temperature. The organics were then extract with ether, washed with 0.5% NaHCO 3 , water, brine and then dried over MgSO 4 . The crude was then dry loaded onto silica and purified by column chromatography using silica gel as the stationary phase and an eluting gradient of 3-5 % ether:petroleum ether to yield a mixture of the four 2-and 4-methyl sterol isomers as a colorless solid (240 mg, 54%), purified by reversed phase HPLC eluted with methanol.
References. As shown in this figure there is a growing understanding of the initial appearance of 2-methyl hopanes in anaerobic prokaryotes that is followed by the appearance of 24-desalkyl/24-alkyl steranes, 4-methyl-steranes and 24/26-methyl steranes in eukaryotes following the advent of atmospheric oxygen. As a working hypothesis based on our sterol evolution and sterol methylation research (see also Supplementary Figure Biochem. 256, 86-96 (1998)) in which two distinct SMT enzyme types were described based on differences in their amino acid sequences that correlated to the substrate properties of the cloned enzymes shown to catalyze the first (SMT1-cycloartenol substrate) and second (SMT2-24(28)-methylene lophenol substrate) methylations of the ∆ 24 -bond, respectively. It is important for evolutionary considerations that thus far, two SMT families of SMT1 and SMT2 provide the foundation for all PHYLOGENOMIC/PHYLOGENETIC papers dealing with sterol methyltransferases.
Two mechanisms for the successive methylation of the sterol side chain can occur: One is based on the view the land plant SMT2 and its surrogates can be substrate promiscuous as in lessadvanced organisms giving rise to compounds 17A and 18A. The other is based on our work of product specificity determined by partitioning-explicit outcomes to ∆ 24(28) -∆ 24(25) -or ∆ 25(27)olefin. Although, we have characterized a "SMT1" from protozoa that generates the ∆ 25(27)sterol chain capable of C27-methyl extension, further C27-sterol side chain modification was not possible due to regio-specificity in the sterol methylation reaction. Thus, the complex side chains of dinoflagellates and marine invertebrate Porifera necessitate a distinct SMT with an evolved active site of appropriate volume to accept SAM additions that can elongate the side chain at C27.