formycin and 5--methylthioadenosine

Topics: Adenine; Catalytic Domain; Crystallography, X-Ray; Deoxyadenosines; Formycins; Helicobacter pylori; Hydrogen Bonding; Models, Molecular; Neutrons; Protein Binding; Protons; Purine-Nucleoside Phosphorylase; Pyrrolidines; S-Adenosylhomocysteine; Substrate Specificity; Thionucleosides

5'-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5'-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5'-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 A resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5'-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5'-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK(a) of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.

Topics: Amino Acid Sequence; Arabidopsis Proteins; Binding Sites; Catalysis; Crystallography, X-Ray; Deoxyadenosines; Hydrolysis; Kinetics; Molecular Sequence Data; Protein Conformation; Purine-Nucleoside Phosphorylase; Substrate Specificity; Thionucleosides

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) and plays a key role in four metabolic processes: biological methylation, polyamine biosynthesis, methionine recycling and bacterial quorum sensing. The absence of the nucleosidase in mammalian species has implicated this enzyme as a target for antimicrobial drug design. MTAN from the pathogenic bacterium Staphylococcus aureus (SaMTAN) has been kinetically characterized and its structure has been determined in complex with the transition-state analogue formycin A (FMA) at 1.7 A resolution. A comparison of the SaMTAN-FMA complex with available Escherichia coli MTAN structures shows strong conservation of the overall structure and in particular of the active site. The presence of an extra water molecule, which forms a hydrogen bond to the O4' atom of formycin A in the active site of SaMTAN, produces electron withdrawal from the ribosyl group and may explain the lower catalytic efficiency that SaMTAN exhibits when metabolizing MTA and SAH relative to the E. coli enzyme. The implications of this structure for broad-based antibiotic design are discussed.

Topics: Amino Acid Sequence; Binding Sites; Catalysis; Crystallization; Deoxyadenosines; Drug Design; Enzyme Inhibitors; Escherichia coli; Formycins; Hydrogen Bonding; Kinetics; Molecular Sequence Data; N-Glycosyl Hydrolases; Protein Binding; Protein Conformation; S-Adenosylhomocysteine; Sequence Homology, Amino Acid; Staphylococcus aureus; Thionucleosides

5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is a key enzyme in a number of critical biological processes in many microbes. This nucleosidase catalyzes the irreversible hydrolysis of the N(9)-C(1') bond of MTA or AdoHcy to form adenine and the corresponding thioribose. The key role of the MTA/AdoHcy nucleosidase in biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing has made it an important antimicrobial drug target. The crystal structures of Escherichia coli MTA/AdoHcy nucleosidase complexed with the transition state analog, formycin A (FMA), and the nonhydrolyzable substrate analog, 5'-methylthiotubercidin (MTT) have been solved to 2.2- and 2.0-A resolution, respectively. These are the first MTA/AdoHcy nucleosidase structures to be solved in the presence of inhibitors. These structures clearly identify the residues involved in substrate binding and catalysis in the active site. Comparisons of the inhibitor complexes to the adenine-bound MTA/AdoHcy nucleosidase (Lee, J. E., Cornell, K. A., Riscoe, M. K., and Howell, P. L. (2001) Structure (Camb.) 9, 941-953) structure provide evidence for a ligand-induced conformational change in the active site and the substrate preference of the enzyme. The enzymatic mechanism has been re-examined.

Topics: Amino Acid Sequence; Catalysis; Deoxyadenosines; Enzyme Inhibitors; Escherichia coli; Models, Molecular; Molecular Sequence Data; N-Glycosyl Hydrolases; Protein Conformation; Sequence Homology, Amino Acid; Substrate Specificity; Thionucleosides