文摘
5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is important in a numberof cellular functions such as polyamine biosynthesis, methionine salvaging, biological methylation, andquorum sensing. The nucleosidase is found in many microbes but not in mammalian systems, thus makingMTAN a broad-spectrum antimicrobial drug target. Substrate binding and catalytic residues were identifiedfrom the crystal structure of MTAN complexed with 5'-methylthiotubercidin [Lee, J. E., Cornell, K. A.,Riscoe, M. K. and Howell, P. L. (2003) J. Biol. Chem. 278 (10) 8761-8770]. The roles of active siteresidues Met9, Glu12, Ile50, Ser76, Val102, Phe105, Tyr107, Phe151, Met173, Glu174, Arg193, Ser196,Asp197, and Phe207 have been investigated by site-directed mutagenesis and steady-state kinetics.Mutagenesis of residues Glu12, Glu174, and Asp197 completely abolished activity. The location of Asp197and Glu12 in the active site is consistent with their having a direct role in enzyme catalysis. Glu174 issuggested to be involved in catalysis by stabilizing the transition state positive charge at the O3', C2', andC3' atoms and by polarizing the 3'-hydroxyl to aid in the flow of electrons to the electron withdrawingpurine base. This represents the first indication of the importance of the 3'-hydroxyl in the stabilizationof the transition state. Furthermore, mutation of Arg193 to alanine shows that the nucleophilic water isable to direct its attack without assistance from the enzyme. This mutagenesis study has allowed areevaluation of the catalytic mechanism.
