Structure of the Month: September 2009 [see all]

Crystal structures of Mycobacterium tuberculosis KasA, a drug target involved in fatty acid biosynthesis

Sylvia R. Luckner, Carl A. Machutta, Peter J. Tonge & Caroline Kisker

Mycobacterium tuberculosis is the organism responsible for tuberculosis (TB) and the disease currently causes more than 2 million deaths worldwide every year (WHO 2007). Novel chemotherapeutics are urgently needed to treat sensitive strains as well as the increasing problem of multi-drug resistant strains. We use structure based drug design to develop novel inhibitors that target essential enzymes of Mycobacterium tuberculosis.

The β-ketoacyl synthase KasA is a key enzyme in the fatty acid biosynthesis pathway II (FASII) of Mycobacterium tuberculosis and a validated drug target. As the structural difference to the fatty acid synthesis in mammals is considerable, it is possible to develop drugs that selectively target bacterial enzymes without affecting the human system. KasA catalyzes the condensation reaction between acyl-ACP and malonyl-ACP and thereby adds two carbon atoms to the growing fatty acid that is elongated in mycobacteria to up to 56 carbon atoms in length. These fatty acids belong to the longest fatty acids found in nature and are essential components of the mycobacterial cell wall (Bhatt, Molle et al. 2007).

The crystal structures of KasA in its wild-type form and the C171Q KasA variant that mimics the acylated form of the enzyme give insight into the molecular mechanism of action of the enzyme. These structures together with the inhibitor bound structures (Luckner, Machutta et al. 2009) serve as a foundation for the design of novel inhibitors that target KasA.

Crystals of the wild-type form of KasA and the C171Q variant were obtained with different crystallization conditions resulting in different crystal forms and different space groups. Datasets of both crystals could be collected at high resolution with the high intensity MicroMax-007 HF X-ray generator. An example of the diffraction pattern is shown in Figure 1B. Crystals from the same setups did not lead to datasets with significantly improved data quality or resolution when the data were collected at the synchrotron.

Comparison of wild-type KasA with the C171Q KasA variant shows that rearrangements occur in the active site upon mutation (or accordingly upon acylation): The side chain of Phe404 is rotated in C171Q KasA to an 'open' conformation and the flexible loop is shifted outside the pocket and creates a larger binding pocket. In addition, the entrance to the pocket is wider in the mutant compared to the wild type enzyme. These findings explain why the binding of inhibitors is facilitated in the acylated enzyme and the C171Q variant.

Long polyethylene glycol (PEG) molecules are bound to the acyl channel of the C171Q KasA structures and mimic a fatty acid of ~40 carbons in length. Thus, the acyl channel and the substrate binding mode of long chain fatty acids can be analyzed. We propose that a scissors-like movement of two gate segments presumably accomplishes binding of the very long chain fatty acids to KasA.

This work was published in Structure 17, 1004-1013, July 15, 2009

Data collection details

References

• Bhatt, A., V. Molle, et al. (2007). "The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development." Mol Microbiol 64(6): 1442-54.

• Luckner, S. R., C. A. Machutta, et al. (2009). "Crystal structures of Mycobacterium tuberculosis KasA show mode of action within cell wall biosynthesis and its inhibition by thiolactomycin." Structure 17(7): 1004-13.

• WHO (2007). "World Health Organization Global Tuberculosis Control: surveillance, planning, financing." WHO Report.