title: RlmN and AtsB as models for the overproduction and characterization of radical SAM proteins.
abstract: An explosion of remarkable chemical transformations has been witnessed in the past decade as a result of the radical S-adenosyl-l-methionine (SAM) (RS) superfamily of proteins. These proteins share the ability to cleave SAM reductively to l-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA(•)). The 5'-dA(•) initiates >40 distinct reaction types by abstracting target hydrogen atoms on small-molecule and macromolecular substrates. All RS enzymes contain a [4Fe-4S] cluster coordinated by SAM that supplies the electron for SAM cleavage. A subset of RS enzymes contains additional iron-sulfur (Fe/S) clusters that serve alternative purposes, many remaining to be defined. The oxygen lability of their [4Fe-4S] clusters causes RS enzymes to be more tedious to purify, characterize, and study. Moreover, the type(s) and stoichiometry of Fe/S clusters in RS enzymes has often been a source of debate. Herein, we use RlmN and AtsB as models to highlight methods for purifying and characterizing RS enzymes, focusing on using Mössbauer spectroscopy in concert with methods for quantifying iron and acid-labile sulfide to assign cluster content accurately.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Lanz
authors.forename: Tyler L
authors.initials: TL
authors.lastname: Grove
authors.forename: Camelia Baleanu
authors.initials: CB
authors.lastname: Gogonea
authors.forename: Kyung-Hoon
authors.initials: KH
authors.lastname: Lee
authors.forename: Carsten
authors.initials: C
authors.lastname: Krebs
authors.forename: Squire J
authors.initials: SJ
authors.lastname: Booker
mesh_terms: D003001:Cloning, Molecular; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism*; D015204:Spectroscopy, Mossbauer
chemical_list: C471554:AtsB protein, Klebsiella pneumoniae; D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D005609:Free Radicals; D007506:Iron-Sulfur Proteins; C033714:5'-deoxyadenosine; D012436:S-Adenosylmethionine; D008715:Methionine; D007501:Iron; D008780:Methyltransferases; C558134:RlmN protein, E coli