Cloning, functional expression, and characterization of Acanthamoeba castellanii and Naegleria gruberi C-24 sterol methyltransferase

A set of sterol methyl transferases (SMT) from amoeba, 2 synthesized in Acanthamoeba castellanii (Ac), and 1 in Naegleria gruberi (Ng), were cloned, functionally expressed in Escherichia coli and their substrate specificities determined. Bioinformatic analysis of the amino acid sequences for the protozoan-distinct SMT’s revealed that they possessed relatedness more to plants than to fungi. Enzyme-generated substrate and product specificities were determined by a combination of GC/MS, NMR, HPLC, and isotopic labelling with D3-SAM. The favored substrate for Ac24SMT was cycloartenol, Ac-28SMT was 24-methylenelophenol, and Ng24SMT was zymosterol, which corresponds to their natural distribution in the respective amoebas. Ac24SMT and Ng24SMT catalyzed sterol methylation through the Δ24(28) pathway affording a single product. Alternatively, Ac28SMT catalyzed sterol methylation through the Δ25(27) pathway, affording 4α-methyl-24βethyl-stigmasta-7,25(27)-dienol and other minor products. Inhibition of the three SMTs was demonstrated using high-affinity transition state analogs (TSA) of the sterol methylation reaction; the TSA were equally potent against the Ac and Ng SMTs and exhibited Ki’s in the low nanomolar range, consistent with their mechanism of action. These results show that a heretofore unrecognized substrate preference and product outcome of SMT catalyzed reactions that could influence ergosterol biosynthesis and growth of these parasitic organisms. SMT is a clear target enzyme that warrants further study in rational drug design targeting pathogenic amoeba.