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We have refined the X-ray structures of two site-directed mutants of the iron-dependent superoxide dismutase (SOD) from Mycobacterium tuberculosis. These mutations which affect residue 145 in the enzyme (H145Q and H145E) were designed to alter its metal-ion specificity. This residue is either Gln or His in homologous SOD enzymes and has previously been shown to play a role in active-site interactions since its side-chain helps to coordinate the metal ion via a solvent molecule which is thought to be a hydroxide ion. The mutations were based on the observation that in the closely homologous manganese dependent SOD from Mycobacterium leprae, the only significant difference from the M. tuberculosis SOD within 10 Å of the metal-binding site is the substitution of Gln for His at position 145. Hence an H145Q mutant of the M. tuberculosis (TB) SOD was engineered to investigate this residue's role in metal ion dependence and an isosteric H145E mutant was also expressed. The X-ray structures of the H145Q and H145E mutants have been solved at resolutions of 4.0 Å and 2.5 Å , respectively, confirming that neither mutation has any gross effects on the conformation of the enzyme or the structure of the active site. The residue substitutions are accommodated in the enzyme's three-dimensional structure by small local conformational changes. Peroxide inhibition experiments and atomic absorption spectroscopy establish surprisingly the H145E mutant SOD has manganese bound to it whereas the H145Q mutant SOD retains iron as the active-site metal. This alteration in metal specificity may reflect on the preference of manganese ions for anionic ligands.Keywords : superoxide dismutase; Mycobacterium tuberculosis; X-ray structure; site-directed mutagenesis.The Mycobacterium tuberculosis superoxide dismutase at an approximate distance of 18 Å . The structure of the M. tu-(SOD) is a 207-residue enzyme with a subunit molecular mass berculosis SOD has recently been determined at 2.0-Å resolution of 23 kDa . It is one of a homologous series establishing that it is a compact tetramer. of superoxide dismutases with Aϩβ tertiary structure which have A diagram of the tertiary structure of the M. tuberculosis SOD either iron or manganese located at the catalytic centre and can is shown in Fig. 1 where the metal-binding site is indicated. be either dimeric or tetrameric. X-ray structures for several other Comparison of the dimer-dimer contacts within the tetrameric members of this family are available: human (Borgstahl et al., members of this family show that they vary greatly from enzyme 1992), Bacillus stearothermophilus (Parker and Blake, 1988a), to enzyme (Wagner et al., 1993;Cooper et al., 1995) giving rise Escherichia coli (Lah et al., 1995), Pseudomonas ovalis (Stod-to a range of quaternary structures despite strong conservation dard et al., 1990) and Thermus thermophilus (Ludwig et al., of the dimer structure. The active-site residues of Fe-SOD and 1991). These enzymes are structurally distinct from the Cu-Zn Mn-SOD are almost completely ...

Since 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU) has been shown to be a potent anti-HBV agent in vitro, it was of interest to study the structure-activity relationships of related nucleosides. Thus, a series of 1-(2-deoxy-2-fluoro-beta-L-arabinofuranosyl)pyrimidine nucleosides have been synthesized and evaluated for antiviral activity against HBV in 2.2.15 cells. For this study, L-ribose was initially used as the starting material. Due to the commercial cost of L-ribose, we have developed an efficient procedure for the preparation of L-ribose derivative 6. Starting from L-xylose, 6 was obtained in an excellent total yield (70%) through the pyridinium dichromate oxidation of the 3-OH group followed by stereoselective reduction with NaBH4. It was further converted to the 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-alpha-L-arabinofuranose (10), which was then condensed with various 5-substituted pyrimidine bases to give the nucleosides. Among the compounds synthesized, the lead compound, L-FMAU (13), exhibited the most potent anti-HBV activity (EC50 0.1 microM). None of the other uracil derivatives showed significant anti-HBV activity up to 10 microM. Among the cytosine analogues, the cytosine (27) and 5-iodocytosine (35) derivatives showed moderately potent anti-HBV activity (EC50 1.4 and 5 microM, respectively). The cytotoxicity of these nucleoside analogues has also been assessed in 2.2.15 cells as well as CEM cells. None of these compounds displayed any toxicity up to 200 microM in 2.2.15 cells. Thus, compound 13 (L-FMAU), 27, and 35 showed a selectivity of over 2000, 140, and 40, respectively.

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