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Substrate Specificity and Unusual Reaction Mechanism of the Sterol 4‐Methyltransferase in Caenorhabdtis elegans
Author(s) -
Fisher Paxtyn Michael,
Zhou Wenxu,
Miller Matthew B.,
Shen Yun,
Shi Huazhong,
Nes W. David
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.629.22
Subject(s) - lanosterol , sterol , cholestanol , methylation , lathosterol , biosynthesis , methyltransferase , biochemistry , oxysterol , chemistry , cholesterol , isomerase , stereochemistry , enol , substrate (aquarium) , desmosterol , enzyme , biology , campesterol , ecology , gene , catalysis
Studies on the biosynthesis of sterols in animals have been the subject of Nobel Prizes. In vertebrates, cholesterol biosynthesis proceeds from lanosterol to sterol intermediates methylated at the 4‐postion (Nes, W. D. (2011) Chem. Rev. 111 , 6423–6451). These compounds typically contain a double bond at the 8‐position and originate by the removal of C4‐methyl from lanosterol. In contrast, the invertebrate worm C. elegans converts cholesterol to C4‐methylsterol end products that possess a double ‐bond at the 7‐ or 8(14)‐position. The C4‐methyl addition is catalyzed by sterol C4‐methyltransferase (4‐SMT) (Hannich, J. T. et al. (2009) Dev. Cell 16 , 833–843). There is speculation that C4‐methyl sterols are produced through a route involving direct methylation of lathosterol (cholest‐7‐enol) to form lophenol (4‐methyl cholest‐7‐enol). There is support for this proposal as C3‐methyl hopanoids are considered to be formed by a radical mechanism of direct methylation. We report here that C. elegans , a sterol auxotroph that converts dietary cholesterol or cholestanol to 4‐methyl sterols, when fed cholest‐3‐one (I) or cholest‐7‐3‐one (II) convert these nutrients to 4‐methyl cholestanol and 4‐methyl cholest‐7‐enol, respectively, by a hypothetical 2‐step metabolism involving a 4‐SMT and 4‐reductase enzyme. Using a cloned 4‐SMT functionally expressed in E. coli and an assay system developed for the sterol C24‐methyltransferase (24‐SMT) that includes SAM or D3‐SAM, we observed that I converts to 4‐methyl cholest‐3‐one while II converts to C4‐methyl cholest‐7‐one or C4‐D3‐methyl‐cholest‐7‐one, respectively. Product identities were confirmed by GC‐MS analysis and synthesis. The results show the 4‐SMT has a broad substrate requirement and reaction of lathosterone (II) in the presence of SAM generates a 3‐keto‐4‐enol intermediate that is regio‐ and stereo‐specifically methylated at C4 affording lophenone, a 4α (equatorial)‐methyl 3‐keto sterol. Notably, the amino acid sequences of 4‐SMT and 24‐SMT share approximate 30 % identity and the 4‐SMT contains all the substrate binding segments for sterol and SAM identified in 24‐SMT. Alternatively, a bioinformatic search for a radical SAM motif identified in bacteria genomes fails to uncover an equivalent binding site in 4‐SMT. Thus, we conclude that the reaction catalyzed by C. elegans 4‐SMT is phyla‐specific and proceeds by a tightly coupled keto‐enol‐methylation mechanism which is different from the reaction catalyzed by 24‐SMT that proceeds by a coupled methyl addition‐proton elimination mechanism.