分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: Fatty acid degradation protein D32 (FadD32), an enzyme required for mycolic acid biosynthesis and essential for mycobacterial growth, has recently been identified as a valid and promising target for anti-tuberculosis drug development. Here we report the crystal structures of Mycobacterium smegmatis FadD32 in the apo and ATP-bound states at 2.4 angstrom and 2.25 angstrom resolution, respectively. FadD32 consists of two globular domains connected by a flexible linker. ATP binds in a cleft at the interface between the N- and C-terminal domains and its binding induces significant local conformational changes in FadD32. The binding sites of meromycolic acid and phosphopantetheine are identified by structural comparison with other members of the adenylating enzyme superfamily. These results will improve our understanding of the catalytic mechanism of FadD32 and help in the design of inhibitors of this essential enzyme.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-05
摘要: DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an alpha beta(2)epsilon polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its a subunit, Mtb DNA pol III has two potential proofreading subunits; the alpha and epsilon subunits. During DNA replication, the presence of the beta(2) clamp strongly promotes the polymerization of the alpha beta(2)epsilon replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb.