Structural and functional studies of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase.
Published: 2005
Language: eng
Number of pages: 208
Description: 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolysis of the ribosidic bond of 5'-methylthioadenosine and S-adenosylhomocysteine to adenine and its corresponding thioribose. The key role of MTAN in methionine recycling, biological methylation, polyamine biosynthesis, and autoinducer-2 quorum sensing biosynthesis has made it an important antimicrobial target.  E. coli MTAN has been over-expressed, purified, and crystallized. Selenomethionine incorporation and the MAD technique were used to determine the initial MTAN crystal structure to 2.4 A resolution. The structure was subsequently refined to 1.9 A resolution using a native sulfur dataset. Each monomer of the MTAN dimer consists of a mixed alpha/beta domain with a nine-stranded mixed beta-sheet, flanked by six alpha-helices and a small 310 helix. In addition, the structures of MTAN complexed to the inhibitors, formycin A and 5'-methylthiotubercidin din were solved. These MTAN-inhibitor structures identified residues important in catalysis and binding. Site-directed mutagenesis and kinetic experiments subsequently confirmed their active site roles. Structures of the apo-enzyme, enzyme-substrate, and enzyme-product complexes were also determined. These structures in combination with the MTAN-inhibitor complexes provide snapshots along the reaction coordinate. This "catalytic movie" suggests a substrate-induced enzyme conformational change and substantial conformational flexibility of the nucleoside substrate during catalysis.To advance our drug design efforts, Immucillin and DADMe-Immucillin purine nucleoside phosphorylase transition state inhibitors were modified to better match the transition state features on MTAN. These inhibitors have binding affinities on the order of 10-12 to 10-14 M and are among the tightest ever described. Structures of MTAN complexed to the parent compounds, 5'-methylthio-Immucillin A and 5'-methylthio-DADMe-Immucillin A were solved. These inhibitor-complexed structures provide extraordinary insight into the structural basis for the tight affinities and will serve as valuable templates for future drug development. Although these inhibitors have high affinities to MTAN, drug design is complicated by their tight binding to human MTA phosphorylase. Structures of human MTA phosphorylase complexed with formycin A and 5'-methylthiotubercidin were determined. Comparisons to their MTAN counterparts reveal key differences in the ribose and 5'-alkylthio binding sites that could be exploited to design MTAN specific inhibitors. Collectively, the work presented in this thesis provides a detailed understanding of the structure and function of MTAN and offers a blueprint for future inhibitor development and structural investigations.
Notes: Source: Dissertation Abstracts International, Volume: 66-10, Section: B, page: 5383.

Thesis (Ph.D.)--University of Toronto, 2005.

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