Acta Cryst. (1999). D55, 865±868 Gill et al.  Glutamine synthetase 865 crystallization papers Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Preliminary crystallographic studies on glutamine synthetase from Mycobacterium tuberculosis Harindarpal S. Gill, Gaston M.U. P¯uegl and David Eisenberg* UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, Departments of Chemistry and Biochemistry and Biological Chemistry, Box 951570, University of California Los Angeles, Los Angeles, CA 90095-1570, USA Correspondence e-mail: david@mbi.ucla.edu # 1999 International Union of Crystallography Printed in Denmark ± all rights reserved The etiologic agent of tuberculosis, Mycobacterium tuberculosis, has been shown to secrete the enzyme glutamine synthetase (TB-GS) which is apparently essential for infection. Four crystal forms of a recombinant TB-GS were grown. The one chosen for synchrotron X-ray data collection belongs to space group P2 1 2 1 2 1 with unit-cell dimensions 208  258  274 A Ê , yielding 2.4 A Ê resolution data. A Matthews number of 2.89 A Ê 3 Da 1 is found, corresponding to 24 subunits of molecular mass 1300 kDa in the asymmetric unit. From earlier work, the structure of Salmonella typhimurium GS, which is 51% identical in sequence to TB-GS, is known to be dodecameric with 622 symmetry. Self-rotation calculations on the TB-GS X-ray data reveal only one set of sixfold and twofold axes of symmetry. A Patterson map calculated from the native X-ray data con®rms that there are two dodecamers in the asymmetric unit, having both their sixfold and twofold axes parallel to one another. Received 15 September 1998 Accepted 15 December 1998 1. Introduction Tuberculosis (TB) is the leading cause of death by a single infectious agent, each year emer- ging as an active disease in eight million humans and causing three million deaths (Kochi, 1991). Existing antimycobacterial drugs are becoming less effective because of the rise in resistant strains (Centers for Disease Control, 1991). Discovery of novel drug targets is complicated by the need to penetrate the thick cellular envelope of mycobacteria. However, pathogenic mycobacteria may have an Achilles heel: they secrete enzymes into their local environment, which may be essen- tial for growth (see Young et al., 1990, for a review). One enzyme of particular interest is glutamine synthetase (TB-GS), product of the glnA gene from M. tuberculosis (Harth et al., 1994). TB-GS is secreted into the medium, where it is thought to play a role in the synthesis of poly-(l-glutamine-l-glutamate) chains (Harth et al., 1994), a constituent unique to pathogenic mycobacterial cell walls, which comprise 10% of the bacterial mass (Hirsch- ®eld et al., 1990). These chains have been reported to be tightly associated with the peptidoglycan layer of the cell wall. Thus, inhibition of secreted TB-GS may disrupt normal cell-wall development and conse- quently the organism's growth, suggesting that secreted TB-GS is an attractive target for TB- speci®c drugs. In microorganisms, glutamine synthetase (GS) is typically a cytoplasmic enzyme central to nitrogen metabolism, catalyzing the ATP- dependent condensation of ammonia and glutamate to form ADP, glutamine and free phosphate (Woolfolk et al., 1966). The X-ray crystal structure of GS from Salmonella typhimurium has been reported at 3.5 A Ê reso- lution (Almassy et al. , 1986) and subsequently re®ned to 2.9 A Ê (Liaw & Eisenberg, 1994). S. typhimurium GS molecules are dodecamers, having two face-to-face hexameric rings, with 12 symmetrically related active sites, one formed between every pair of subunits within a ring. Protein-sequence alignments show a 51% identity between S. typhimurium GS and TB-GS, suggesting a common tertiary fold for the two molecules. As the ®rst step in the Figure 1 Crystals of recombinant TB-GS. Two crystal forms of rTB-GS grew in a single crystallization drop. The multi- sided chunky crystals belong to space group P2 1 2 1 2 1 (form 2) and the rod-shaped elongated crystals can belong either to space group P2 1 2 1 2 1 or space group P2 1 (form 2 or 1). Dimensions vary from 0.5 to 1 mm in each dimension.