title: Molecular architecture of TylM1 from Streptomyces fradiae: an N,N-dimethyltransferase involved in the production of dTDP-D-mycaminose.
abstract: d-Mycaminose is an unusual dideoxy sugar found attached to the antibiotic tylosin, a commonly used veterinarian therapeutic. It is synthesized by the Gram-positive bacterium Streptomyces fradiae as a dTDP-linked sugar. The last step in its biosynthesis involves the dimethylation of the hexose C-3' amino group by an S-adenosylmethionine (SAM) dependent enzyme referred to as TylM1. Here we report two high-resolution X-ray structures of TylM1, one in which the enzyme contains bound SAM and dTDP-phenol and the second in which the protein is complexed with S-adenosylhomocysteine (SAH) and dTDP-3-amino-3,6-dideoxyglucose, its natural substrate. Combined, these two structures, solved to 1.35 and 1.79 Å resolution, respectively, show the orientations of SAM and the dTDP-linked sugar substrate within the active site region. Specifically, the C-3' amino group of the hexose is in the correct position for an in-line attack at the reactive methyl group of SAM. Both Tyr 14 and Arg 241 serve to anchor the dTDP-linked sugar to the protein. To test the role of His 123 in catalysis, two site-directed mutant proteins were constructed, H123A and H123N. Both mutant proteins retained catalytic activity, albeit with reduced rates. Specifically, the k(cat)/K(m) was reduced to 1.8% and 0.37% for the H123A and H123N mutant proteins, respectively. High-resolution X-ray models showed that the observed perturbations in the kinetic constants were not due to major changes in their three-dimensional folds. Most likely the proton on the C-3' amino group is transferred to one of the water molecules lining the active site pocket as catalysis proceeds.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Carney
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Holden
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013942:Thymine Nucleotides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013942:Thymine Nucleotides; C013610:mycaminose; D008780:Methyltransferases; D005944:Glucosamine