Zhihong Song

Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols.

Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s–1, respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-l-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. The high energy intermediate analog 25-azacycloartanol was a noncompetitive inhibitor versus cycloartenol and an uncompetitive inhibitor versus AdoMet. The dead end inhibitor analog cyclolaudenol was competitive versus cycloartenol and uncompetitive versus AdoMet. 24(28)-Methylenecycloartanol and AdoHcy generated competitive and noncompetitive kinetic patterns, respectively, with respect to AdoMet. Therefore, 24(28)-methylenecycloartanol combines with the same enzyme form as does cycloartenol and must be released from the enzyme before AdoHcy. 25-Azacycloartanol inhibited the first and second C1 transfer activities with about equal efficacy (Ki = 45 nm), suggesting that the successive C-methylation of the Δ24 bond occurs at the same active center. Comparison of the initial velocity data using AdoMet versus [2H3-methyl]AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on VCH3/VCD3 close to unity. [25-2H]24(28)-Methylenecycloartanol, [28E-2H]24 (28)-methylenelanosterol, and [28Z-2H]24(28)-methylene lanosterol were prepared and paired with AdoMet or [methyl-3H3]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the Δ24 bond.

Sterol Biosynthesis Inhibitors: Potential for Transition State Analogs and Mechanism-Based Inactivators Targeted at Sterol Methyltransferase

Sterol biosynthesis inhibitors (SBIs), discovered in the late 1960s and subsequently used commercially to treat ergosterol-dependent fungal diseases, represent a unique drug class targeted at an enzyme in a biosynthetic pathway. To date, few drugs have been commercialized as enzyme inhibitors; yet, prescription of SBIs has emerged as the gold standard for some cases of non-life-threatening antifungal chemotherapy and in crop protection. SBIs are not designed for their structural resemblance to the sterol molecule; they nonetheless can engender a curative effect by interfering with sterol production and homeostasis in the pathogenic organism. The increased use of SBIs in recent years, particularly the azole antifungals, has resulted in the development of resistance to those drugs, necessitating additional work to further our understanding of antifungal resistance and to explore opportunities to develop new enzyme inhibitors and uncover new enzyme targets that can regulate carbon flux in the post-lanosterol/cycloartenol pathway. This article reports general considerations for enzyme mechanism and active-site probes using inhibitors of the C-methylation reaction, including a potential new class of antifungal/antiparasitic agents of phytosterol synthesis tailored as mechanism-based inactivators. These steroid-based compounds prepared with different sterol side chain functionalities are designed to reversibly or irreversibly impair the sterol methyltransferase, an enzyme expressed in pathogenic microbes and plants but not in the human host. The salient aspects of these and related topics directed toward the enzyme recognition of sterol structure, and the inhibitory properties and catalytic competence of a series of specifically modified substrate analogs that affect sterol methyltransferase action are discussed.

Cloning, functional expression and phylogenetic analysis of plant sterol 24C-methyltransferases involved in sitosterol biosynthesis

Sterol 24C-methyltransferases (SMTs) constitute a group of sequence-related proteins that catalyze the distinct patterns of 24-alkyl sterols that occur throughout nature. Two SMT cDNAs (SMT2-1 and SMT2-2) were cloned by homology based PCR methods from young leaves of Glycine max (soybean) and the corresponding enzymes were expressed functionally in Escherichia coli. The full-length cDNA for SMT2-1 and SMT2-2 have open reading frames of 1086 bp and 1092 bp, respectively, and encode proteins of 361 and 363 residues with a calculated molecular mass of 40.3 and 40.4 kDa, respectively. The substrate preference of the two isoforms was similar yet they differed from SMT1; kinetically SMT2-1 and SMT2-2 generated k(cat) values for the optimal substrate 24(28)methylene lophenol of 0.8 min(-1) and 1.34 min(-1), respectively, compared to the activity of SMT1 that generated a k(cat) for the optimal substrate cycloartenol of 0.6 min(-1). SMT2-2 was purified to homogeneity and the subunit organization shown to be tetrameric in similar fashion to other cloned SMTs. Analysis of the accumulated products catalyzed by the recombinant enzymes demonstrated that soybean SMT2-1 and SMT2-2 operate transalkylation activities analogous to the soybean plant SMT1. Metabolite analyses correlated with transcript profiling of the three SMT isoforms during soybean maturation clearly demonstrated that SMT isoform expression determines specific C24-methyl to C24-ethyl ratios to flowering whereas with seed development there is a disconnection such that the SMT transcript levels decrease against an increase in sterol content; generally SMT2-2 is expressed more than SMT2-1 or SMT1. These observations suggest that the genes that encode SMT1 and SMT2 in sitosterol biosynthesis may have undergone divergent evolution. In support of this proposition, the genomic organization for SMT1 of fungi and protozoa align very closely with one another and to those of the plant SMT2; both sets of SMTs lack introns. Unexpectedly, the SMT1 from Glycine max and other embryophytes of diverse origin possess disparate intron-exon characteristics that can be shown relates back to the algae. Our results suggest that the order of SMT1 appearing before SMT2 in phytosterol synthesis arose recently in plant evolution in response to duplication of a more primitive SMT gene likely to have been bifunctional and catalytically promiscuous.

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

Current progress on the mechanism and substrate recognition by sterol methyl transferase (SMT), the role of mechanism-based inactivators, other inhibitors of SMT action to probe catalysis and phytosterol synthesis is reported. SMT is a membrane-bound enzyme which catalyzes the coupled C-methylation-deprotonation reaction of sterol acceptor molecules generating the 24-alkyl sterol side chains of fungal ergosterol and plant sitosterol. This C-methylation step can be rate-limiting in the post-lanosterol (fungal) or post-cycloartenol (plant) pathways. A series of sterol analogs designed to impair SMT activity irreversibly have provided deep insight into the C-methylation reaction and topography of the SMT active site and as reviewed provide leads for the development of antifungal agents.