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Lessons From the Studies of a CC Bond Forming Radical SAM Enzyme in Molybdenum Cofactor Biosynthesis.

Publication ,  Journal Article
Pang, H; Yokoyama, K
Published in: Methods Enzymol
2018

MoaA is one of the founding members of the radical S-adenosyl-L-methionine (SAM) superfamily, and together with the second enzyme, MoaC, catalyzes the construction of the pyranopterin backbone structure of the molybdenum cofactor (Moco). However, the exact functions of both MoaA and MoaC had remained ambiguous for more than 2 decades. Recently, their functions were finally elucidated through successful characterization of the MoaA product as 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP), which was shown to be converted to cyclic pyranopterin monophosphate (cPMP) by MoaC. 3',8-cH2GTP was produced in a small quantity and was highly oxygen sensitive, which explains why this compound had previously eluded characterization. This chapter describes the methodologies for the characterization of MoaA, MoaC, and 3',8-cH2GTP, which together significantly altered the view of the mechanism of the pyranopterin backbone construction during the Moco biosynthesis. Through this chapter, we hope to share not only the protocols to study the first step of Moco biosynthesis but also the lessons we learned from the characterization of the chemically labile biosynthetic intermediate, which would be informative for the study of many other metabolic pathways and enzymes.

Duke Scholars

Published In

Methods Enzymol

DOI

EISSN

1557-7988

Publication Date

2018

Volume

606

Start / End Page

485 / 522

Location

United States

Related Subject Headings

  • Recombinant Proteins
  • Pterins
  • Pteridines
  • Organophosphorus Compounds
  • Molybdenum Cofactors
  • Metalloproteins
  • Metabolic Networks and Pathways
  • Hydrolases
  • Escherichia coli Proteins
  • Enzyme Assays
 

Citation

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Pang, H., & Yokoyama, K. (2018). Lessons From the Studies of a CC Bond Forming Radical SAM Enzyme in Molybdenum Cofactor Biosynthesis. Methods Enzymol, 606, 485–522. https://doi.org/10.1016/bs.mie.2018.04.014
Pang, Haoran, and Kenichi Yokoyama. “Lessons From the Studies of a CC Bond Forming Radical SAM Enzyme in Molybdenum Cofactor Biosynthesis.Methods Enzymol 606 (2018): 485–522. https://doi.org/10.1016/bs.mie.2018.04.014.
Pang, Haoran, and Kenichi Yokoyama. “Lessons From the Studies of a CC Bond Forming Radical SAM Enzyme in Molybdenum Cofactor Biosynthesis.Methods Enzymol, vol. 606, 2018, pp. 485–522. Pubmed, doi:10.1016/bs.mie.2018.04.014.

Published In

Methods Enzymol

DOI

EISSN

1557-7988

Publication Date

2018

Volume

606

Start / End Page

485 / 522

Location

United States

Related Subject Headings

  • Recombinant Proteins
  • Pterins
  • Pteridines
  • Organophosphorus Compounds
  • Molybdenum Cofactors
  • Metalloproteins
  • Metabolic Networks and Pathways
  • Hydrolases
  • Escherichia coli Proteins
  • Enzyme Assays