Production and Characterization of Class C Radical Sam Methylases Nosn and Blmorf8

Open Access
Author:
Gadsby, Lauren Kathryn
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Squire J Booker, Thesis Supervisor
  • David Scott Gilmour, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • SAM
  • iron-sulfur cluster
  • radical chemistry
  • methylase
  • NosN
  • BlmOrf8
  • nosiheptide
  • bleomycin
  • S-adenosyl-methionine
Abstract:
Essential in the maintenance of gene expression, metabolism, and the modification of macromolecules, methyl transfer reactions often involve S-adenosyl-methionine (SAM) as the methyl donor and are catalyzed by a group of enzymes known as SAM-dependent methylases. While some enzymes employ an SN2 mechanism to methylate their substrates, a subset of the methylases have been shown to employ radical chemistry and a 5’-deoxyadenosyl 5’-radical (5’dA•) intermediate to catalyze methylation. These radical-dependent methylases have been grouped into three classes—A, B, and C—that are separated according to structural and mechanistic similarities. All members of the RS superfamily are distinguished by certain structural features, including the presence of at least one [4Fe-4S] cluster that is coordinated by the Cys residues of a conserved CxxxCxxC motif. While members of Class A RS methylases have been moderately characterized, very little mechanistic insight is available for Class B enzymes, which appear to use methylcobalamin as a cosubstrate, and even less is known about Class C enzymes. Representative Class C enzymes NosN and BlmOrf8, involved in the biosynthesis of the antibiotics nosiheptide and bleomycin, respectively, have been identified based on conserved elements in their amino acid sequences. In this study, NosN and BlmOrf8 were heterologously overproduced in Escherichia coli, purified, reconstituted, and characterized using analytical and spectroscopic methods. Characterization of, and investigation into, the mechanisms of NosN and BlmOrf8 have revealed that these enzymes contain one [4Fe-4S] cluster thought to bind SAM and aid in cleavage to S-adenosyl-homocysteine (SAH) and 5’dA•. This study suggests that, for the representative enzyme NosN, the enzyme binds two equivalents of SAM simultaneously to assemble the newly introduced methyl group.