Yu, Hong (2007-05). Structural studies of Mycobacterium tuberculosis KatG, an INH drug activator, and Brucella abortus VirB11, an ATPase of type IV translocation system. Doctoral Dissertation. | Thesis individual record

Catalase-peroxidase (KatG) of Mycobacterium tuberculosis is a bifunctional heme
enzyme that has been shown to play an important role in the activation of a first line
drug, isoniazid (INH), used in the treatment of tuberculosis infection. Mutations in the
katG gene have been found to be associated with INH resistance. The most commonly
encountered mutation is the Ser315Thr point mutation. In this dissertation, the x-ray
crystallographic structures of MtbKatG and the mutant enzyme KatG[S315T] are
presented to explore the molecular basis of the INH activation and resistance. The
structure is dimeric and contains a heme cofactor in each subunit of the dimer. The most
important change in KatG[S315T] is due to the presence of the methyl group of the
threonine 315 side chain, which is located at the narrowest part of the substrate channel.
The protruding methyl group effectively constricts the accessibility to the heme by
closing down the dimensions of the channel, constraining the substrate entrance.
VirB11 of Brucella abortus is a hexameric ATPase that belongs to the type IV
secretion system. The crystal structure of BaVirB11 was found to contain six molecules
per asymmetric unit. The Walker A (P loop), His box, and Glu box from the C-terminal domain are located at the interface of the N- and C-terminal domain. A large
conformational change was found in the linker region when compared with that of
HP0525 structure, the VirB11 analogous from H. pylori. To elucidate the functional role
of each domain, seven functional mutations were generated and used for biochemical
studies. The GER motif and the linker region were found to be crucial for ATP
hydrolysis activity of BaVirB11. Mutations in the GER motif (R101Q) and the linker
region (R120E) of BaVirB11 completely abolish the ATP hydrolysis activity of the
enzyme. The binding affinities of the two mutants to the ATP; however, are similar to
that of the wild-type enzyme, indicating that mutation in the GER motif or the linker
region has no effect on ATP binding.

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