Biochemical and Structural Characterisation of a Thermostable Cfr-Like Enzyme from Sphaerobacter thermophilus

Abstract

Cfr is a radical SAM (S-adenosyl-L-methionine) methyltransferase capable of conferring resistance to phenicols, linocosmides, oxazolidinones, pleuromutilins and streptogramin A (termed the PhLOPSA phenotype) via the C8 methylation of 23s rRNA residue A2503. Radical SAM enzymes utilise a [4Fe-4S] cluster to coordinate the reductive cleavage of SAM producing a 5’Deoxyadenosyl radical, which is used to catalyse reactions at inherently unreactive carbon centres. To date, attempts to crystallise Cfr have been unsuccessful and all structural data originate from RlmN, a Cfr homolog with no resistance phenotype capable of methylating position C2 of the same residue.

To address the void of structural knowledge regarding Cfr, four Cfr-like enzymes were selected for investigation into their biochemical characteristics and potential for crystallisation: the Cfr(B) and Cfr(C) alleles originally identified in resistant pathogens, and the PbCfr (Paenibacillus spp.) and SbCfr (Sphaerobacter thermophilus) homologues from thermophilic organisms. Work presented here shows expression of SbCfr and Cfr in recombinant E. coli BL21* and anaerobic purification by immobilised metal ion chromatography, yielding 10.25 and 22.4 mg.L-1 E. coli culture, respectively. UV-vis spectra of SbCfr display a 420nm peak consistent with the presence of intact [4Fe-4S] clusters before and after reconstitution, while anaerobic activity assays show s-adenosylhomocysteine (SAH) and 5’ deoxyadenosine (DOA) formation by both reconstituted and unreconstituted SbCfr. Activity assays and circular dichroism spectroscopy indicate SbCfr activity up to 55ºC and stability up to 65ºC. However, SbCfr did not confer the PhLOPSA phenotype on recombinant E. coli, while efforts to use site-directed mutants to trap covalent RNA complexes, indicative of RlmN-like activity, were similarly unsuccessful.

Although crystallisation trials of Cfr were not successful, SbCfr was successfully crystallised and the structure determined to 1.4 A resolution, revealing a monomeric enzyme containing a partial TIM β-barrel, hosting a [4Fe-4S] cluster coordinated by the canonical radical SAM enzyme CX3CX2C motif, and resembling previous structures of E. coli RlmN. The structure contains an SAH ligand bound to the 4Fe-4S cluster and a methyl group attached to Cys334, which adopts a conformation resembling that observed in RNA-bound RlmN but distinct from that in complexes with SAM. This represents a state not previously described in RlmN structures. This work shows SbCfr to be a stable radical SAM enzyme with likely methyltransferase activity but that is not associated with antimicrobial resistance and suggests that conformational changes previously thought to occur on RNA binding occur earlier in the reaction cycle of radical SAM methyltransferases.

Date of Award	1 Oct 2019
Original language	English
Awarding Institution	University of Bristol
Supervisor	Matthew B Avison (Supervisor) & Jim Spencer (Supervisor)