PubMed · 6067906
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J Gressel, E Galun. 1967. Morphogenesis in Trichoderma: photoinduction and RNA.. https://doi.org/10.1016/0012-1606(67)90054-1
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Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine-salvage pathway, which allows cells to utilize preformed bases and nucleosides in order to synthesize nucleotides. PNP is specific for purine nucleosides in the beta-configuration and exhibits a strong preference for purines containing a 6-keto group and ribosyl-containing nucleosides relative to the corresponding analogues. PNP was crystallized in complex with ligands and data collection was performed using synchrotron radiation. This work reports the structure of human PNP in complex with guanosine (at 2.80 A resolution), 3'-deoxyguanosine (at 2.86 A resolution) and 8-azaguanine (at 2.85 A resolution). These structures were compared with the PNP-guanine, PNP-inosine and PNP-immucillin-H complexes solved previously.
BACKGROUND: Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase. RESULTS: The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 micromol/min/mg protein and the Km for methionine and ATP was 288 microM and 76 microM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 microM, while the MIC for 8-azaguanine was >1.0 mM. CONCLUSION: The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors.
Watson-Crick optimized geometries and the energies of base pairing for the natural pairs of nucleic bases: adenine-thymine (AT) and guanine-cytosine (GC) have been recalculated by ab initio methods in order to compare results to those found for the non-natural azaadenine-thymine (AAT) and azaguanine-cytosine (AGC) pairs. Geometry optimizations carried out at the HF/6-31G** level and energies obtained at MP2/6-31G**, show that AAT and AGC have hydrogen bonding patterns similar to the natural AT and GC and that the interaction energies (DeltaH0int) for the former are ca. 7 kcal/mol more stable than the latter. Accordingly, the pairs based on azapurines would be favored with respect to the natural pairs. Some possible explanations why nature does not use extensively the azabases in base pairing are given.