Structural and Functional Characterization of CalS11, a TDP-Rhamnose 3′-<i>O</i>-Methyltransferase Involved in Calicheamicin Biosynthesis

Singh, Shanteri; Chang, Andrew S.; Helmich, K.E.; Bingman, C.A.; Wrobel, Russell L.; Beebe, Emily T.; Makino, Shin-ichi; Aceti, David J.; Dyer, Kevin; Hura, Greg L.; Sunkara, Manjula; Morris, Andrew J.; Phillips, George N.; Thorson, Jon S.

doi:10.1021/cb400068k

Cited by 12 publications

(15 citation statements)

References 41 publications

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“…MycF has an active site similar to those of other metal-dependent O -methyltransferases (OMT), including catechol OMT 31 , alfalfa caffeoyl coenzyme A 3-OMT 32 , the TDP-rhamnose 3-OMT CalS11 28 , the novobiocin 4′-OMT NovP 23 , and the mycinamicin 2′-OMT MycE 20 . These metal-dependent OMTs share a conserved SAM binding site, metal binding site and catalytic base position (Lys in catechol OMT and caffeoyl CoA OMT, His in MycE, and Asp in CalS11, NovP and MycF.)…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2015

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2015

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“…In contrast with sugar C‐ or N‐methylation, which typically occurs at the sugar nucleotide stage prior to attachment of the sugar to the aglycone, most sugar‐O‐methylation occurs after the sugar has been transferred to the core aglycone . However, two sugar‐O‐methyltransferases, CalS and AvrH, derived from the calicheamicin and avermectin biosynthetic pathways, respectively, have been reported to methylate sugars at the nucleotide stage . To determine the reaction sequence of AlmCII‐catalyzed 3′‐O‐methylation, a feeding experiment was first carried out by incubating compound 7 with the previously established PKS‐inactivated strain JHZ1001‐2, and the products were analyzed by HPLC.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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Sugar O-methylation is a ubiquitous modification in natural products and plays diverse roles. This realization has inspired many attempts to search for novel methyltransferases. Chalcomycins are a group of 16-membered macrolides containing two methylated sugars that require three methyltransferases for their biosynthesis. Here, we identified that AlmCII, a sugar O-methyltransferase belonging to the TylF family that was previously only known to methylate sugars with a 4'-hydroxy group, can methylate a 4',6'-dideoxysugar during the biosynthesis of chalcomycins. An in vitro enzymatic assay revealed that AlmCII is divalent metal-dependent with an optimal pH of 8.0 and optimal temperature of 42 °C. Moreover, the 3'-O-demethylated chalcomycins exhibit less than 6 % of the antibacterial activity of their parent compounds. This is the first report demonstrating that a TylF family O-methyltransferase can use a 4'-deoxy sugar as a substrate and highlighting the importance of this methylation for the antibacterial activity of chalcomycins.

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“… 21,22 CalS11, a protein encoded by the calicheamicin biosynthetic gene locus, 23,24 catalyzes a late-stage glycosyl tailoring event (thymidine diphosphate (TDP)-L-rhamnose 3′- O -methylation) prior to glycosyltransferase (CalG1)-catalyzed transfer to complete aryltetrasaccharide assembly (Figure 1 ). 10,25 Like all prototypical class I methyltransferases, 26 CalS11 uses S -adenosylmethionine (AdoMet, SAM) as the methyl donor. However, CalS11 is distinguished from other sugar O -methyltransferases by virtue of its activity at the sugar nucleotide prior to glycosyltransfer.…”

Section: Introductionmentioning

confidence: 99%

“…However, CalS11 is distinguished from other sugar O -methyltransferases by virtue of its activity at the sugar nucleotide prior to glycosyltransfer. 10,27 …”

Section: Introductionmentioning

confidence: 99%

Loop dynamics of thymidine diphosphate-rhamnose 3′-O-methyltransferase (CalS11), an enzyme in calicheamicin biosynthesis

et al. 2016

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Structure analysis and ensemble refinement of the apo-structure of thymidine diphosphate (TDP)-rhamnose 3′-O-methyltransferase reveal a gate for substrate entry and product release. TDP-rhamnose 3′-O-methyltransferase (CalS11) catalyses a 3′-O-methylation of TDP-rhamnose, an intermediate in the biosynthesis of enediyne antitumor antibiotic calicheamicin. CalS11 operates at the sugar nucleotide stage prior to glycosylation step. Here, we present the crystal structure of the apo form of CalS11 at 1.89 Å resolution. We propose that the L2 loop functions as a gate facilitating and/or providing specificity for substrate entry or promoting product release. Ensemble refinement analysis slightly improves the crystallographic refinement statistics and furthermore provides a compelling way to visualize the dynamic model of loop L2, supporting the understanding of its proposed role in catalysis.

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Structural and Functional Characterization of CalS11, a TDP-Rhamnose 3′-O-Methyltransferase Involved in Calicheamicin Biosynthesis

Cited by 12 publications

References 41 publications

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

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

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

Loop dynamics of thymidine diphosphate-rhamnose 3′-O-methyltransferase (CalS11), an enzyme in calicheamicin biosynthesis

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