The Crystal Structure of TrpD, a Metabolic Enzyme Essential for Lung Colonization by Mycobacterium tuberculosis, in Complex with its Substrate Phosphoribosylpyrophosphate Clare E. Lee 1 , Catherine Goodfellow 1 , Farah Javid-Majd 2 Edward N. Baker 1 and J. Shaun Lott 1 * 1 Laboratory of Structural Biology and Centre for Molecular Biodiscovery, School of Biological Sciences University of Auckland, Private Bag 92019, Auckland 1020 New Zealand 2 Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU College Station TX 77843-2128, USA Mycobacterium tuberculosis, the cause of tuberculosis, presents a major threat to human health worldwide. Biosynthetic enzymes that are essential for the survival of the bacterium, especially in activated macrophages, are important potential drug targets. Although the tryptophan biosynthesis pathway is thought to be non-essential for many pathogens, this appears not to be the case for M. tuberculosis, where a trpD gene knockout fails to cause disease in mice. We therefore chose the product of the trpD gene, anthranilate phosphoribosyltransferase, which catalyses the second step in tryptophan biosynthesis, for structural analysis. The structure of TrpD from M. tuberculosis was solved by X-ray crystallography, at 1.9 A ˚ resolution for the native enzyme (RZ0.191, R free Z0.230) and at 2.3 A ˚ resolution for the complex with its substrate phosphoribosylpyrophosphate (PRPP) and Mg 2C (RZ0.194, R free Z0.255). The enzyme is folded into two domains, separated by a hinge region. PRPP binds in the C-terminal domain, together with a pair of Mg ions. In the substrate complex, two flexible loops change conformation compared with the apo protein, to close over the PRPP and to complete an extensive network of hydrogen-bonded interactions. A nearby pocket, adjacent to the hinge region, is postulated by in silico docking as the binding site for anthranilate. A bound molecule of benzamidine, which was essential for crystallization and is also found in the hinge region, appears to reduce flexibility between the two domains. q 2005 Elsevier Ltd. All rights reserved. Keywords: tuberculosis; tryptophan biosynthesis; anthranilate phospho- ribosyltransferase; substrate binding; crystal structure *Corresponding author Introduction Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains the single most devasta- ting human pathogen, currently causing an esti- mated 5000 deaths per day worldwide. 1 Although protracted, antibiotic therapy is cheap and can be very effective in treating the disease, with high cure rates among compliant patients. However, current therapy is being undermined by a number of factors, most significantly the phenomenon of persistence (whereby a proportion of the bacteria remains unaffected by current drugs), the emer- gence of multiple drug resistant (MDR) strains of M. tuberculosis, and co-infection with human immunodeficiency virus (HIV). The treatment of MDR strains of M. tuberculosis is expensive and relatively ineffective, particularly so in immune- compromised patients, such as HIV-infected indi- viduals. One major goal for the development of new therapies is therefore the development of cheap and efficacious antibiotics for more effective treatment of persistent infections and MDR-TB. Tuberculosis is an airborne infection, and once inhaled into the lung, the bacterium is phagocytosed 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: TB, tuberculosis; MDR, multiple drug resistant; HIV, human immunodeficiency virus; PRPP, phosphoribosylpyrophosphate; ORF, open reading frame; PDB, Protein Data Bank; SeMet, selenomethionine; MAD, multi-wavelength anomalous diffraction; RMSD, root-mean-square difference. E-mail address of the corresponding author: s.lott@auckland.ac.nz doi:10.1016/j.jmb.2005.11.016 J. Mol. Biol. (2006) 355, 784–797