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Title: 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 definemore » the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Authors:
 [1];  [2];  [2];  [2];  [2];  [3];  [2];  [3];  [4];  [5]
  1. Univ. of Michigan, Ann Arbor, MI (United States); Univ. of Michigan, Ann Arbor, MI (United States). Life Sciences Inst.
  2. Univ. of Michigan, Ann Arbor, MI (United States). Life Sciences Inst.
  3. Toho Univ., Funabashi, Chiba (Japan)
  4. Univ. of Michigan, Ann Arbor, MI (United States). Life Sciences Inst.; Univ. of Michigan, Ann Arbor, MI (United States). Dept. of Medicinal Chemistry, Chemistry, and Microbiology & Immunology
  5. Univ. of Michigan, Ann Arbor, MI (United States). Life Sciences Inst.; Univ. of Michigan, Ann Arbor, MI (United States). Dept. of Biological Chemistry
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Institutes of Health (NIH)
OSTI Identifier:
1213729
Grant/Contract Number:  
AC02-06CH11357; 5T32GM008597; DK042303; GM078553
Resource Type:
Accepted Manuscript
Journal Name:
ACS Chemical Biology
Additional Journal Information:
Journal Volume: 10; Journal Issue: 5; Journal ID: ISSN 1554-8929
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Bernard, Steffen M., Akey, David L., Tripathi, Ashootosh, Park, Sung Ryeol, Konwerski, Jamie R., Anzai, Yojiro, Li, Shengying, Kato, Fumio, Sherman, David H., and Smith, Janet L. Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.. United States: N. p., 2015. Web. doi:10.1021/cb5009348.
Bernard, Steffen M., Akey, David L., Tripathi, Ashootosh, Park, Sung Ryeol, Konwerski, Jamie R., Anzai, Yojiro, Li, Shengying, Kato, Fumio, Sherman, David H., & Smith, Janet L. Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.. United States. doi:10.1021/cb5009348.
Bernard, Steffen M., Akey, David L., Tripathi, Ashootosh, Park, Sung Ryeol, Konwerski, Jamie R., Anzai, Yojiro, Li, Shengying, Kato, Fumio, Sherman, David H., and Smith, Janet L. Wed . "Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.". United States. doi:10.1021/cb5009348. https://www.osti.gov/servlets/purl/1213729.
@article{osti_1213729,
title = {Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.},
author = {Bernard, Steffen M. and Akey, David L. and Tripathi, Ashootosh and Park, Sung Ryeol and Konwerski, Jamie R. and Anzai, Yojiro and Li, Shengying and Kato, Fumio and Sherman, David H. and Smith, Janet L.},
abstractNote = {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. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.},
doi = {10.1021/cb5009348},
journal = {ACS Chemical Biology},
number = 5,
volume = 10,
place = {United States},
year = {2015},
month = {2}
}

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