title: Glutamate 2,3-aminomutase: a new member of the radical SAM superfamily of enzymes.
abstract: A gene eam in Clostridium difficile encodes a protein that is homologous to lysine 2,3-aminomutase (LAM) in many other species but does not have the lysyl-binding residues Asp293 and Asp330 in LAM from Clostridium subterminale SB4. The C. difficile protein has Lys and Asn, respectively, in the sequence positions of the essential Asp residues in LAM. The C. difficile gene has been cloned into an E. coli expression vector, expressed in E. coli, and the protein purified and characterized. The recombinant protein displays excellent activity as a glutamate 2,3-aminomutase and no activity toward l-lysine. The PLP-, iron-, and sulfide-content and ultraviolet/visible spectrum are similar to LAM, and the enzyme requires SAM and dithionite as activators, as does LAM. Freeze-quench EPR experiments in the presence of l-glutamate reveal a glutamate-based free radical in the steady state of the reaction. A number of other bacterial genomes include genes encoding proteins homologous to the glutamate 2,3-aminomutase from C. difficile, and four of these proteins display the activity of glutamate 2,3-aminomutase when produced in E. coli. All of the homologous proteins have the cysteine motif CSMYCRHC corresponding to the motif CxxxCxxC characteristic of radical SAM enzymes. It is concluded that glutamate 2,3-aminomutase from C. difficile is a representative of a family found in a number of bacteria. It is likely that the beta-glutamate found in a few bacterial and archeal species as an osmolyte arises from the action of glutamate 2,3-aminomutase.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, 1710 University Avenue, Madison, WI 53726, USA.
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Ruzicka
authors.forename: Perry A
authors.initials: PA
authors.lastname: Frey
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003013:Clostridium / Q000201:enzymology; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D019751:Intramolecular Transferases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D019751:Intramolecular Transferases; C518333:glutamate 2,3-aminomutase, Clostridium difficile