Structure of a critical metabolic enzyme:S-adenosylmethionine synthetase fromCryptosporidium parvum

Abstract

S-Adenosyl-l-methionine (AdoMet), the primary methyl donor in most biological methylation reactions, is produced from ATP and methionine in a multistep reaction catalyzed by AdoMet synthetase. The diversity of grouptransfer reactions that involve AdoMet places this compound at a key crossroads in amino-acid, nucleic acid and lipid metabolism, and disruption of its synthesis has adverse consequences for all forms of life. The family of AdoMet synthetases is highly conserved, and structures of this enzyme have been determined from organisms ranging from bacteria to humans. Here, the structure of an AdoMet synthetase from the infectious parasite Cryptosporidium parvum has been determined as part of an effort to identify structural differences in this enzyme family that can guide the development of speciesselective inhibitors. This enzyme form has a less extensive subunit interface than some previously determined structures, and contains some key structural differences from the human enzyme in an allosteric site, presenting an opportunity for the design of selective inhibitors against the AdoMet synthetase from this organism.

A growth‐based assay using fluorescent protein emission to screen for S‐adenosylmethionine synthetase inhibitors

“…The FP genes were introduced into the existing pUC57‐MAT plasmids, where the MAT open reading frames were from E. coli (GenBank #NP_417417) or C. parvum (#AA017675), under the control of P BAD . The MAT clones have been described previously (Ohren et al, 2019; Parungao et al, 2017); some were PCR amplified from chromosomal DNA and those not available from this source (or containing introns) were synthesized (GenScript). The expression cassette from pHSG298‐FP (donor vector), containing the FP gene under P lac , was then integrated into the acceptor vector using Gibson assembly so the final constructs had bicistronic inserts (Figure 1).…”

“…To expand this screening assay for the testing of MAT orthologs from other organisms, the metK growth defect in E. coli MOB1490 has been complemented by MAT genes from nine pathogenic microbes (Parungao et al, 2017). For the present proof‐of‐principle study the MAT gene from the apicomplexan protozoal pathogen Cryptosporidium parvum was selected (Ohren et al, 2019), along with the metK (MAT) gene from E. coli to serve as a positive control for complementation. Each of the complementing plasmids rescue the growth of the ∆ metK E. coli strain without the need for additional AdoMet supplementation (Parungao et al, 2017).…”