Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of Rv0241c (HtdX) from Mycobacterium tuberculosis H37Rv

Abstract

Rv0241c (HtdX) is a putative (3R)-hydroxyacyl-CoA dehydratase of Mycobacterium tuberculosis. The htdX gene belongs to a conserved operon and is expressed in mycobacteria in the presence of several fatty-acid synthase II drugs. To elucidate the structure of HtdX, the protein was cloned, overexpressed, purified to homogeneity and crystallized. The protein was crystallized from two conditions: (i) 3 M sodium chloride, 0.1 M Na HEPES pH 8.0 and (ii) 2.5 M sodium chloride, 0.1 M Tris-HCl pH 8.5. A complete diffraction data set was collected from crystals from both conditions. The crystal from the first condition diffracted to 2.3 Å resolution and belonged to space group I41, with unit-cell parameters a=b=61.51, c=143.81 Å. Crystals from the second condition diffracted to 3.1 Å resolution and belonged to space group P4₃2₁2 or P4₁2₁2, with unit-cell parameters a=b=63.67, c=140.88 Å. Both crystals contained one molecule in the asymmetric unit.

Keywords: Mycobacterium tuberculosis; Rv0241c (HtdX).

Figure 1

Crystals of HtdX. (a) Crystals obtained from 3 M sodium chloride, 0.1 M Na HEPES pH 8. (b) Crystals obtained from 2.5 M sodium chloride, 0.1 M Tris–HCl pH 8.5.

Figure 2

Diffraction images collected from crystals from (a) condition (i) and (b) condition (ii). For each frame, the oscillation width and the exposure time were 1° and 3 min, respectively.