Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv

Abstract

Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 angstroms, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 angstroms resolution using a synchrotron-radiation source.

Figure 1

SDS–PAGE analysis of recombinant M. tuberculosis prephenate dehydratase protein purification. Lane 1, crude extract; lane 2, BenchMark Ladder protein standards (Invitrogen; 10–220 kDa); lane 3, ammonium sulfate precipation; lane 4, Q-Sepharose Fast Flow anion exchange; lane 5, phenyl Sepharose HP hydrophobic interaction; lane 6, Mono Q HR anion exchange.

Figure 2

Crystal of recombinant M. tuberculosis prephenate dehydratase with approximate dimensions of 0.5 × 0.01 × 0.02 mm.

Figure 3

X-ray diffraction pattern of recombinant M. tuberculosis prephenate dehydratase.