Biochemical characterization of bacterial methyltransferases reveals necessary residues for sterol side-chain propylation

The capacity to methylate sterol side-chains via sterol methyltransferases (SMTs) was thought to be widespread in fungi, algae, and plants but limited in animals and nonexistent in bacteria. We have previously demonstrated that yet-uncultured bacteria have the genomic capacity to produce side-chain methylated sterols de novo. Further, we identified three bacterial SMTs capable of producing 24-isopropyl sterols and showed that each of these SMTs was biochemically sufficient for all three of the side-chain methylation steps necessary for 24-isopropyl sterol synthesis. To date, no eukaryotic SMT has been identified that is capable of sequentially adding three methyl groups to generate this propyl structure. To better understand this unique biochemical feature of bacterial SMTs and to potentially identify key amino acid residues involved in sequential methylations on the sterol side-chain, we performed site-directed mutagenesis of propylating bacterial SMTs. Through these analyses, we identified a glycine residue that is necessary but not sufficient for side-chain propylation. This residue is located outside known SMT substrate-binding domains, but inside the active site of an SMT protein model docked with 24-methylenecholesterol. We also show that phenylalanine residues in sterol-binding Region I increase the production of 24-isopropyl sterols, and that 25 residues are conserved among methylating, ethylating, and propylating SMTs. Together, the presence of these residues may allow us to predict if an organism has the genomic capacity to produce C-24 propylated sterols directly from sequencing data, and to generate hypotheses about the environments in which bacterial sterol side-chain propylation may be occurring through analysis of metagenomes. Given the high preservation potential of side-chain alkylated sterols and their use as molecular fossils indicative of ancient life deep in time, a better understanding of the distribution of these unique lipids in modern life and present-day ecosystems will allow for more robust interpretations of side-chain alkylated steranes in the rock record.