3pfg

Publication Abstract from PubMed

D-Mycaminose is an unusual dideoxysugar 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-A and 1.79-A 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 kcat/Km 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.

Molecular Architecture of TylM1 from Streptomyces fradiae: an N,N-Dimethyltransferase Involved in the Production of dTDP-D-Mycaminose.,Carney AE, Holden HM Biochemistry. 2010 Dec 13. PMID:21142177^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.