Expression, purification and preliminary crystallographic analysis of Rv3002c, the regulatory subunit of acetolactate synthase (IlvH) from Mycobacterium tuberculosis

Abstract

Branched amino-acid biosynthesis is important to bacterial pathogens such as Mycobacterium tuberculosis (Mtb), a microorganism that presently causes more deaths in humans than any other prokaryotic pathogen (http://www.who.int/tb). In this study, the molecular cloning, expression, purification, crystallization and preliminary crystallographic analysis of recombinant IlvH, the small regulatory subunit of acetohydroxylic acid synthase (AHAS) in Mtb, are reported. AHAS carries out the first common reaction in the biosynthesis of valine, leucine and isoleucine. AHAS is an essential enzyme in Mtb and its inactivation leads to a lethal phenotype [Sassetti et al. (2001), Proc. Natl Acad. Sci. USA, 98, 12712-12717]. Thus, inhibitors of AHAS could potentially be developed into novel anti-Mtb therapies.

Figure 1

Sequence alignment of Mtb IlvH and its homologues from E. coli, Nitrosomonas europaea and Thermotoga maritima. The secondary structures corresponding to N. europaea IlvH (PDB entry 2pc6; Petkowski et al., 2007 ▶) are drawn at the top of the sequence alignment. Helices, β-strands and turns are represented by coils, arrows and letter ‘T’s, respectively. Conserved residues are highlighted in red, whereas residues with similar chemical properties are colored red. The sequence alignment was performed using the program ClustalW (Thompson et al., 1994 ▶) and the figure was generated with the program ESPript (Gouet et al., 1999 ▶).

Figure 2

Morphology of Rv3002c crystals. The approximate dimensions of Rv3002c crystals of diffraction quality grown over a period of 3–4 d were 10 × 50 × 200 µm.

Figure 3

A representative frame of the Rv3002c data set showing the diffraction quality. Rings indicate resolutions.