3PFH

X-Ray crystal structure the N,N-dimethyltransferase TylM1 from Streptomyces fradiae in complex with SAH and dTDP-Quip3N

Summary for 3PFH

• Entry DOI: 10.2210/pdb3pfh/pdb
• Related: 3PFG
• Descriptor: N-methyltransferase, S-ADENOSYL-L-HOMOCYSTEINE, [(3R,4S,5S,6R)-4-amino-3,5-dihydroxy-6-methyloxan-2-yl][hydroxy-[[(2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy]phosphoryl] hydrogen phosphate, ... (5 entities in total)
• Functional Keywords: n, n-dimethyltransferase, sam binding, dtdp-linked sugar binding, transferase
• Biological source: Streptomyces fradiae
• Total number of polymer chains: 2
• Total formula weight: 59001.35

Authors

Carney, A.E.,Holden, H.M. (deposition date: 2010-10-28, release date: 2010-12-15, Last modification date: 2023-09-06)

Primary citation

Carney, A.E.,Holden, H.M.
Molecular Architecture of TylM1 from Streptomyces fradiae: An N,N-Dimethyltransferase Involved in the Production of dTDP-d-mycaminose .
Biochemistry, 50:780-787, 2011

PubMed 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.

PubMed: 21142177
DOI: 10.1021/bi101733y

Experimental method

X-RAY DIFFRACTION (1.792 Å)