Abstract
Catechol O-methyltransferase (COMT) is widely distributed in nature and installs a methyl group onto one of the vicinal hydroxyl groups of a catechol derivative. Enzymes belonging to this family require two cofactors for methyl transfer: S-adenosyl-l-methionine as a methyl donor and a divalent metal cation for regiospecific binding and activation of a substrate. We have determined two high-resolution crystal structures of Rv0187, one of three COMT paralogs from Mycobacterium tuberculosis, in the presence and absence of cofactors. The cofactor-bound structure clearly locates strontium ions and S-adenosyl-l-homocysteine in the active site, and together with the complementary structure of the ligand-free form, it suggests conformational dynamics induced by the binding of cofactors. Examination of in vitro activities revealed promiscuous substrate specificity and relaxed regioselectivity against various catechol-like compounds. Unexpectedly, mutation of the proposed catalytic lysine residue did not abolish activity but altered the overall landscape of regiospecific methylation.
Highlights
Catechol O-methyltransferase (COMT) is widely conserved in bacteria, plants, fungi, and metazoa
When the protein construct of Rv0187 fused with a C-terminal His6-tag was analyzed, a minor peak appeared close to the void volume of the column, and a major peak emerged around the elution volume consistent with that of a dimeric form, which is the typical oligomeric state of class I COMTs (Supplementary Fig. 2)
The ratio between high oligomer and the dimer is reversed in the case of an N-terminal His6-tagged sample, where the protein elutes as high-order oligomers more predominantly than in the C-terminal His6-tagged version
Summary
Catechol O-methyltransferase (COMT) is widely conserved in bacteria, plants, fungi, and metazoa. The chemical reaction catalyzed by this family (pfam methyltransf_3, or PF01596) involves the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to a hydroxyl group of catechol-like compounds (Fig. 1) These enzymes are class I O-methyltransferases (OMTs) and typically composed of 200–250 amino acids and require a divalent metal ion for full activity. Twenty crystal structures of bacterial COMTs have been reported from 14 species to date, and eight of them contain a divalent cation in the conserved metal-binding site, e.g., Mg2+, Mn2+, and Ni2+ 11,12,17–19. Detailed cellular functions of these paralogs are still elusive Among these COMTs, the crystal structure of Rv1220 has been determined, which surprisingly displays a lack of the signature metal-binding site conserved in Class I COMTs (Supplementary Fig. 1)[20]. Combined with genetic analysis results, our structural and biochemical data provide valuable insights into the biological function of Rv0187
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
