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

Lu Han,Jon S Thorson,Shanteri Singh,George N Phillips

doi:10.1063/1.4941368

Lu Han, Jon S Thorson + Show 2 more

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https://doi.org/10.1063/1.4941368

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Abstract

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.

Highlights

Structure analysis and ensemble refinement of the apo-structure of thymidine diphosphate (TDP)-rhamnose 30-O-methyltransferase reveal a gate for substrate entry and product release
Ensemble refinement analysis slightly improves the crystallographic refinement statistics and provides a compelling way to visualize the dynamic model of loop L2, supporting the understanding of its proposed role in catalysis
One targeted pathway for this initiative has been that leading to the biosynthesis of calicheamicin (CLM), a 10-membered enediyne antitumor antibiotic produced by Micromonospora echinospora

Summary

Introduction

Natural products remain invaluable sources for drug leads and bioactive probes. Discovering new mechanisms for the biosynthesis of important natural products and exploiting knowledge of natural product biosynthesis enzymes could help produce new diversified biosynthetic or semisynthetic natural products for various purposes. As part of the NIH Protein Structure Initiative, a high-throughput structural genomics approach has been employed to clone, express, purify, and solve structures of novel enzymes for natural product biosynthesis. One targeted pathway for this initiative has been that leading to the biosynthesis of calicheamicin (CLM), a 10-membered enediyne antitumor antibiotic produced by Micromonospora echinospora. Upon bioreduction, CLM undergoes a Bergman-type cyclization reaction, the benzene diradical species of which lead to DNA backbone hydrogen abstraction and subsequent irreparable oxidative DNA strand scission. CalS11, a protein encoded by the calicheamicin biosynthetic gene locus, catalyzes a late-stage glycosyl tailoring event (thymidine diphosphate (TDP)-L-rhamnose 30-O-methylation) prior to glycosyltransferase (CalG1)-catalyzed transfer to complete aryltetrasaccharide assembly (Figure 1). Like all prototypical class I methyltransferases, CalS11 uses S-adenosylmethionine (AdoMet, SAM) as the methyl donor. Natural products remain invaluable sources for drug leads and bioactive probes.. As part of the NIH Protein Structure Initiative, a high-throughput structural genomics approach has been employed to clone, express, purify, and solve structures of novel enzymes for natural product biosynthesis.. One targeted pathway for this initiative has been that leading to the biosynthesis of calicheamicin (CLM), a 10-membered enediyne antitumor antibiotic produced by Micromonospora echinospora.. CalS11, a protein encoded by the calicheamicin biosynthetic gene locus, catalyzes a late-stage glycosyl tailoring event (thymidine diphosphate (TDP)-L-rhamnose 30-O-methylation) prior to glycosyltransferase (CalG1)-catalyzed transfer to complete aryltetrasaccharide assembly (Figure 1).. Like all prototypical class I methyltransferases, CalS11 uses S-adenosylmethionine (AdoMet, SAM) as the methyl donor. CalS11 is distinguished from other sugar O-methyltransferases by virtue of its activity at the sugar nucleotide prior to glycosyltransfer.

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