Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O-Methyltransferases.

Abstract

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6′-deoxyallose substituent occurs in a stepwise manner first at the 2′- and then the 3′-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3′-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2′-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4′-specific homolog, active site residues were identified that correlate with the 3′- or 4′- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.

Biosynthesis of mycobacterial methylmannose polysaccharides requires a unique 1- O -methyltransferase specific for 3- O -methylated mannosides

“…The ribose moiety of the cofactor establishes a bidentate interaction with the side-chain carboxyl of Asp71, closely resembling the contacts observed for Asp122 from NovP and Asp113 from MycF (SI Appendix, Fig. S10 B and C) (30,31). The hydroxyl groups of the ribose engage also in hydrogen bonds with Gly75 and the side chain of Thr100, the latter via a water molecule.…”

“…The Active Site and the Mechanism of Action of MeT1. The closest structural MeT1 homologs are divalent metal-dependent O-MTases (Table 2), often containing a magnesium ion bound at the active site or displaying preference for this metal in activity assays (30,(32)(33)(34)(35). In the SAH-bound MeT1 structure a magnesium ion could be identified, coordinated by the imidazole group of His169, the side-chain carboxylates of the highly conserved Asp170 and Asp141, two solvent molecules, and a molecule of glycerol, in an octahedral arrangement ( Fig.…”

Characterization of Methyltransferase AlmCII in Chalcomycin Biosynthesis: The First TylF Family O‐Methyltransferase Works on a 4′‐Deoxysugar

“…grouped the aligned sequences of 13 experimentally characterized TylF family members according to their substrate structures and methylation sites. They found that two amino acid residues (indicated by a pentacle in Figure S1), Trp58 (NovP number) and Gln246 (MycF number), were highly correlated with the regiospecificity of methyl transfer . Gln246 forms a hydrogen bond with the 4′‐hydroxy group and is conserved among 3′‐OMTs, but is replaced by smaller polar amino acids (Asp or Thr) in 4′‐OMTs.…”

“…In our study, we observed that AlmCII could methylate a 4,6‐dideoxysugar to give chalcose, which is the first example of a TylF member using a sugar without a 4′‐hydroxy group as a substrate. It has been proposed that 3′‐OMT has a preference for substrates with 3′‐ and 4′‐ syn ‐hydroxy groups located on the opposite face of the glycosidic bond, whereas 4′‐OMT requires anti ‐hydroxy groups at the 3′ and 4′ positions and needs the methylation site and glycosidic bond on the same face . Given the essential role of metal ions in maintaining AlmCII catalytic activity, lack of a 4′‐hydroxy in the substrate demethylchalcose would require an additional hydroxy group to serve as a metal ligand, which might be mediated by the 2′‐hydroxy of the sugar ring or a water molecule near the active site.…”

“…Recent crystallographic studies of NovP and MycF revealed that TylF family enzymes possess ac haracteristic N-terminal helical lid domain that gates the substrate access to the SAM binding site. [19] This distinct structural feature has made TylF family enzymes unlikely to accept significant modification at the sugar moieties, but theyc an show increasing substrate promiscuity as af unctiono ft he distance of the modifications from the methylation site. These facts suggest that TylF family enzymes can be used as versatile tools to methylate various glycosylated natural products.…”