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 homologue, 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.

Figure 1

Sugar methylation in natural product biosynthesis. a) Ordered sugar O-methylation in the mycinamicin pathway. MycE catalyzes the 2′-O-methylation of the 6′-deoxyallose of mycinamicin VI 1 to generate the javose of mycinamicin III 2. MycF catalyzes the 3′-O-methylation of javose to form the mycinose of mycinamicin IV 3. The P450 monoxygenase, MycG, catalyzes hydroxylation and epoxide ring formation to produce the final product mycinamicin II 4. b) Substrates for the 4′-O-methytransferase NovP, desmethyldescarbamoyl novobiocin 5, and the 3′-O-methytransferase TylF, macrocin 6. Modified sugars are highlighted in blue, and sites of methylation are denoted with red asterisks.

Figure 2

Structure of MycF. a) MycF monomer with SAH (orange sticks) and Mg²⁺ (magenta sphere) in the active site. b) MycF dimer with N-terminal interface. c-e) SAM binding orders the active site lids and creates a binding pocket for mycinamicin III 2. c) Free enzyme with disordered lid domain and lid loop and a solvent-exposed active site Mg²⁺ (magenta sphere). d) MycF-SAH. The lid domain (yellow) closes over the bound co-substrate, and the lid loop is partially ordered. e) MycF-SAH-substrate. Mycinamicin III (2, green sticks) binding fully orders the lid loop (dark blue). Spheres mark the boundaries of disordered regions. MycF is specific for the javose sugar and makes no specific contacts with the macrolactone ring or desosamine sugar. f) Hydrophobic interactions of MycF with the substrate macrolactone core. View is orthogonal to c–e. g) Substrate binding. Three Asp residues coordinate Mg²⁺. Mg²⁺ coordination of the mycinamicin III 2 3′- and 4′-hydroxy groups position the substrate for catalysis. The SAM methyl group (modeled) is 3.1 Å from the 3′-hydroxy. Asp191 is positioned to act as the catalytic base.

Figure 3

Comparison of MycE and MycF active sites. a) Surface representation. The active site of MycE ( ; 3SSN, left) is at the intersection of three subunits (yellow, green, cyan) whereas the MycF (right; cyan) substrate binds to a single subunit. The MycE and MycF active site lids direct substrate (cyan, green) entry on opposite sides of the catalytic Mg²⁺ (magenta) and SAM (orange), shown in identical orientations. b) MycE (yellow) and MycF (cyan) active sites. The different substrate orientations result in different sugar conformations, sugar coordination of Mg²⁺, and regiochemistry of methyltransfer.

Figure 4

MycF activity with MycE substrate (mycinamicin VI 1). a) MycF active site with 2′-methoxy specificity pocket. Residues selected for substitution experiments are shown in gray sticks. b) HPLC chromatograms of MycF variants with the MycE substrate (mycinamicin VI 1). Wild type MycF and MycF/M56A show production of a new product that has a retention time similar to mycinamicin III 2. c) Mass spectrum of HPLC-purified product of MycF/M56A reaction with the MycE substrate. The M+H peak for the product has a mass comparable to that for the MycE product (monoisotopic mass of mycinamicin III 2 681.4 Da compared to 682.4 Da for the M+H peak). d) Structure of 3'-methoxy-mycinamicin VI 7. COSYand HMBCAD correlations are indicated; NMR spectra are in supplementary information.

Figure 5

Regiochemistry of methyltransfer in MycF homologs. a) Model of NovP substrate complex. The NovP 4′ O-MT is in dark blue, substrate DDN 5 in gray and SAM in orange. b) Structure of MycF-substrate complex with MycF in cyan, mycinamicin III 2 in green and SAM in orange. c) Partial sequence alignment of MycF homologs (MycF numbering). All MycF/TylF family members with known substrates are grouped based on the product sugar, shown at right. Tryptophan at position 49 is strictly conserved among 4′-specific enzymes, as is glutamine at position 246 in 3′-specific enzymes. The conserved tryptophan contributes to the hydrophobic pocket for the 5′-methyl group (shown in a). MycF: mycinamicin 3′-O-methyltransferase , TylF: tylosin 3′-O-methyltransferase , ChmMII: chalcomycin 3′-O-methyltransferase , NovP: novobiocin 4′-O-methyltransferase, CouP: coumermycin 4′-O-methyltransferase , CloP: clorobiocin 4′-O-methyltransferase , BusH: butenyl-spinosyn 4′-O-methyltransferase , SpnH: spinosyn 4′-O-methyltransferase , MtfC: Mycobacterium avium glycopetidolipid 4′-O-methyltransferase, MtfB: Mycobacterium avium glycopetidolipid 4′-O-methyltransferase, Rmt4 Mycobacterium chelonae glycopetidolipid 4′-O-methyltransferase, ElmMIII: elloramycin 4′-O-methyltransferase , SnogL: nogalamycin 4′-O-methyltransferase .