Tryptophan biosynthesis in Mycobacterium tuberculosis

J. Shaun Lott,^a Anthony J. Harrison,^a Emily Parker,^b Rochelle J. Ramsay,^a
and Edward N. Baker^a

^aLaboratory of Structural Biology, School of Biological Sciences, University of Auckland, Private Bag 92-019, Auckland, New Zealand; ^bInstitute of Fundamental Sciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand (s.lott@auckland.ac.nz)

Mycobacterium tuberculosis is the most successful pathogen of mankind. More people are killed by M. tuberculosis infection than by infection with any other bacterium. Recent estimates of the worldwide problem indicate that more than 1000 new cases of tuberculosis (TB) occur every hour, resulting in the death of more than 7000 people per day. Although TB is readily treated with antibiotic therapy, the treatment is slow (6-9 months) and incomplete therapy has given rise to TB strains which are resistant to one or all of the preferred antibiotics [1,2]. The rise of multiple drug resistant (MDR) TB strains has contributed to the increase in the incidence of TB in the major industrialised nations throughout the 1990s, reversing the steady decline of previous decades [3] and has precipitated the search for new therapies and antibiotics.

Tryptophan biosynthesis makes an attractive target for the design of new anti-TB drugs, as auxotrophic mutants of M. tuberculosis are essentially avirulent [4] and the biosynthetic pathway is not present in humans. The first step in tryptophan biosynthesis is carried out by the anthranilate synthase (AS) complex, made up of two polypeptides, trpE and trpG. There are two candidates trpE ORFs in M. tuberculosis (Rv1609 and Rv2386c) but no clear candidate for trpG. The second step in tryptophan biosynthesis is carried out by anthranilate phosphoribosyltransferase, trpD (Rv2192c).

We have expressed in E. coli and purified trpD, trpE and trpE2 from M. tuberculosis as either GST fusion proteins or histidine-tagged proteins. TrpD has been crystallised and data has been collected from these crystals to a resolution of 2.3Å. TrpE2 has also been crystallised and these crystals diffract to a resolution of ~3.5Å. We are currently working to improve the quality of these crystals and to prepare selenomethionine-substituted protein for MAD phasing.

References

1 Rattan, A., Kalia, A. and Ahmad, N. (1998) "Multidrug-resistant Mycobacterium tuberculosis: Molecular Perspectives." Emerging Infectious Diseases 4, 195-209.

2 World Health Organisation (2000) Drug-resistant strains of TB increasing worldwide.

3 Stokstad, E. (2000) "Drug-Resistant TB on The Rise." Science, 287, 2391.

4 Smith, D.A., Parish, T., Stoker, N.G. and Bancroft, G.J. (2001) "Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates." Infection & Immunity 69, 1142-1150.