In certain ways all sterols are the same, yet in other respects, they are quite different. Cholesterol, ergosterol and sitosterol are among the compounds which are not only widely recognized but are also commonly associated with the membranes of animals, fungi/protozoa and plants, respectively. The major difference in the structure of cholesterol with that of ergosterol and sitosterol is at carbon-24 in the sterol side chain; C24 is an H, beta-CH3 or alpha-C2H5 group, respectively. It would appear, therefore, that there is some sort of association between the size and direction of the C24-alkyl group(s) added to the "cholesterol" side chain and that the unique biology of microbes and plants require distinct types and amounts of phytosterols (24-alkyl sterols) to grow and reproduce. Understanding the structure and function of the (S)-adenosyl-L-methionine-delta24-sterol methyltransferase (SMT), the crucial transalkylating enzyme(s) involved in phytosterol biosynthesis and homeostasis, are of significant importance as insights gained from these experiments may facilitate the design of methods to control or mimic their actions.
With previous NSF support, variant SMTs were cloned and characterized and a series of sterol derivatives with modified side chains were investigated as mechanistic probes for this enzyme. A family of structurally similar enzymes was discovered to exist in nature, which catalyzes the successive C-methylations of a sterol acceptor molecule from a common active site according to a similar mechanistic plan (the steric-electric plug model of sterol C24-alkylation). Through site-directed mutagenesis experiments, three of four substrate binding regions in the active site topography were identified to affect the complement of C1/C2-activities and product diversity. Now, in this project, SMTs with novel properties will be created by a combination of rational design and activity assay. In addition, the properties of SMTs from different phyla across kingdoms will be determined. Thermodynamic analysis that determines the entropic as well as enthalpic components of the C24-methylation reaction toward substrates and inhibitors of this reaction type will be conducted; this information will be correlated to the enzyme-generated product distributions. The location of active site residues in cloned SMTs and an estimation of the active SMT enzyme concentration in cell-free preparations of different organisms at different times in their life history will be determined utilizing protein chemistry and X-ray crystallography utilizing synthetic fluorinated analogs and mechanism-based inactivators to afford phyla-specific information on phytosterol biosynthesis.
Broader Impacts This project brings together a number of different efforts centered on phytosterol and SMT structure, biochemistry and evolution. This project presents an opportunity for cross-talk of scientific concepts between undergraduate and graduate students with the postdoctoral associates and PI. Knowledge gained from the SMT characterization and redesign experiments will define relevant topographies in the active center of SMTs to affect catalysis. The hallmark of enzyme evolution that relates to the energetics of enzyme efficiency (the lowering of activation barriers to promote catalytic superiority of the extant enzyme over the ancestral form) will be addressed for the first time by studying phylogenetically different SMTs. The project will give rise to other broader impacts through the education of students by providing them mentoring and experimental approaches used routinely in biochemistry, molecular biology and the study of natural products.
Intellectual Merit: Sterol C24-methyltransferases (SMTs) constitute a group of sequence-related proteins that are absent from vertebrates while catalyzing the pattern of sterol diversity in eukaryote organisms. These essential catalysts produce a structural feature in the form of a 24-alkyl group in the phytosterol side chain which is crucial to growth and maturation of plants as well as parasitic micro-organisms responsible for variant animal and plant diseases. For this NSF-funded project, we characterized the properties of a range of SMTs across kingdoms and studied their functional contribution to phytosterol biosynthesis using mechanism-based inhibitors of SMT catalysis and by tracking 2H- and 13C-labeled intermediates to final products with sensitive labeling techniques. The major findings are: (i) new evolutionarily conserved 24-alkyl sterol biosynthesis pathways to ergosterol in green algae and trypanosomatid protozoa have been elucidated which are distinct from the ergosterol biosynthesis pathway in fungi; (ii) ancient and promiscuous SMT2 from green algae was shown to undergo mutational divergence and a change in substrate specificity and reaction channeling to generate the more-advanced SMT2 of vascular plants involved with C28-methylation of obtusifoliol. These phyla-specific substrate preferences of SMT enzymes and changes in C24-methylation pathway outcomes correspond to the switch in ergosterol biosynthesis to stigmasterol biosynthesis in the green lineage; (iii) Inhibition of protozoan and fungal SMT1 that prefer zymosterol as substrate blocks sterol methylation in micro-organisms. As a result of this inhibition, the micro-organisms will not be able to form ergosterol and cannot survive; and (iv) genetic engineering soybean plants with a combination of foreign HMGCoA-reductase gene linked to soybean SMT1 and SMT2 genes led to improved seed traits. These observations suggest that the change in primordial SMT structure into more modern forms could affect the modifications in phytosterol composition that contribute to the evolution of superior membrane systems necessary for the advancement of plant life. Broader Impacts: Studies carried out during the course of the NSF-funded project led to 20 refereed papers in high impact journals, including Chemical Reviews, numerous presentations of the research by group members at national and international meetings, the PI mentoring 7 undergraduate students, including those from under-represented groups, 9 graduate students, and 3 post-doctoral and visiting scientists. All the undergraduate students are co-authors of the published work and they either matriculated to medical school or graduate school. The graduate students who received MS or PhD degrees moved to industry or post-doctoral and academic positions. The genetic, enzymic and chemical outline of new biosynthesis pathways to ergosterol have been incorporated into community-led biochemical pathway databases for protozoa and algae. Additionally, the genetic materials and compounds produced in these studies have been made available to the scientific community. Based on the insights gained from this work, we expect further research on SMTs may facilitate the design of methods to control or mimic their actions to improve seed quality and provide new antifungal or antiprotozoan leads to treat animals or plants infected with pathogenic organisms.
