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Background: 5'-methylthioadenosine/S-adenosyl-homocysteine (MTA/AdoHcy) nucleosidase catalyzes the irreversible cleavage of 5'-methylthioadenosine and S-adenosylhomocysteine to adenine and the corresponding thioribose, 5'-methylthioribose and S-ribosylhomocysteine, respectively. While this enzyme is crucial for the metabolism of AdoHcy and MTA nucleosides in many prokaryotic and lower eukaryotic organisms, it is absent in mammalian cells. This metabolic difference represents an exploitable target for rational drug design., Results: The crystal structure of E. coli MTA/AdoHcy nucleosidase was determined at 1.90 A resolution with the multiwavelength anomalous diffraction (MAD) technique. Each monomer of the MTA/AdoHcy nucleosidase dimer consists of a mixed alpha/beta domain with a nine-stranded mixed beta sheet, flanked by six alpha helices and a small 3(10) helix. Intersubunit contacts between the two monomers present in the asymmetric unit are mediated primarily by helix-helix and helix-loop hydrophobic interactions. The unexpected presence of an adenine molecule in the active site of the enzyme has allowed the identification of both substrate binding and potential catalytic amino acid residues., Conclusions: Although the sequence of E. coli MTA/AdoHcy nucleosidase has almost no identity with any known enzyme, its tertiary structure is similar to both the mammalian (trimeric) and prokaryotic (hexameric) purine nucleoside phosphorylases. The structure provides evidence that this protein is functional as a dimer and that the dual specificity for MTA and AdoHcy results from the truncation of a helix. The structure of MTA/AdoHcy nucleosidase is the first structure of a prokaryotic nucleoside N-ribohydrolase specific for 6-aminopurines.

A recombinant form of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (E.C. 3.2.2.9) has been purified to homogeneity and crystallized using the hanging-drop vapour-diffusion technique. While several different crystallization conditions were obtained, only one set of conditions yielded crystals suitable for X-ray diffraction analysis. These crystals grow as diamond-shaped wedges, with unit-cell parameters a = 50.92, b = 133.99, c = 70.88 A, alpha = beta = gamma = 90 degrees. The crystals belong to space group P2(1)2(1)2 and diffract to a minimum d spacing of 2.3 A on a MAR345 image plate with a Rigaku RU-200 rotating-anode X-ray generator. On the basis of density calculations, two monomers are predicted per asymmetric unit (Matthews coefficient, V(M) = 2.37 A(3) Da(-1)), with a solvent content of 48%.

The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) is responsible for cleavage of the glycosidic bond in both 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). Based on amino acid sequence analysis of this enzyme from Klebsiella, we recently speculated that an open reading frame found in E. coli (designated pfs) encoded MTA/SAH nucleosidase. To explore this possibility, we amplified, cloned, and expressed the complete pfs gene from E. coli genomic DNA. The recombinant protein exhibited a molecular weight and Michaelis constants for MTA that are in agreement with those reported for native enzyme. From this biochemical evidence we confirm our original assignment of the pfs gene as encoding MTA/SAH nucleosidase.

Recombinant E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) was used to study the potential for this enzyme to serve as a target for chemotherapeutic intervention. An examination of the parameters required for enzymatic activity indicate that the nucleosidase functions over a broad range of pH and temperature, with acidic conditions and temperatures of 37-45 degrees C being optimal. Analogs of 5'-methylthioadenosine and adenosine were assessed as potential enzyme inhibitors and to provide details regarding substrate specificity and reaction mechanism. The 5'-arylthio analog, 5'-(p-nitrophenyl)thioadenosine, was the most potent enzyme inhibitor studied, with a Ki of 20nM. A mutant of the nucleosidase lacking the first 8 amino acids was engineered to determine the contribution of these conserved residues toward enzyme specificity. The truncated enzyme exhibited a K(m)[MTA] of 1.43 microM, approximately 3 fold higher than the K(m) reported for the full-length nucleosidase.

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiella pneumoniae. Chromatography using a novel 5'-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46-50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 microM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 microM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.