Crystal Structure of a Transcarbamylase-like Protein from the Anaerobic Bacterium Bacteroides fragilis at 2.0 A ˚ Resolution Dashuang Shi 1 * , Rene Gallegos 2 , Joseph DePonte III 2 Hiroki Morizono 1 , Xiaolin Yu 1 , Norma M. Allewell 3 , Michael Malamy 2 and Mendel Tuchman 1 1 Children’s Research Institute Children’s National Medical Center, 111 Michigan Avenue N.W., Washington DC 20010-2970, USA 2 Department of Microbiology and Molecular Biology, Tufts University School of Medicine Boston, MA 02111, USA 3 College of Life Science, 2300 Symons Hall, University of Maryland, College Park MD 20742, USA A transcarbamylase-like protein essential for arginine biosynthesis in the anaerobic bacterium Bacteroides fragilis has been purified and crystallized in space group P4 3 2 1 2(a ¼ b ¼ 153.4 A ˚ , c ¼ 94.8 A ˚ ). The structure was solved using a single isomorphous replacement with anomalous scatter- ing (SIRAS) and was refined at 2.0 A ˚ resolution to an R-factor of 20.6% (R-free ¼ 25.2%). The molecular model is trimeric and comprises 960 amino acid residues, two phosphate groups and 422 water molecules. The monomer has the consensus transcarbamylase fold with two structural domains linked by two long interdomain helices: the putative carbamoyl phosphate-binding domain and a binding domain for the second substrate. Each domain has a central parallel b-sheet surrounded by a-helices and loops with a/b topology. The putative carbamoyl phos- phate-binding site is similar to those in ornithine transcarbamylases (OTCases) and aspartate transcarbamylases (ATCases); however, the second substrate-binding site is strikingly different. This site has several insertions and deletions, and residues critical to substrate binding and catalysis in other known transcarbamylases are not conserved. The three- dimensional structure and the fact that this protein is essential for arginine biosynthesis suggest strongly that it is a new member of the trans- carbamylase family. A similar protein has been found in Xylella fastidiosa, a bacterium that infects grapes, citrus and other plants. q 2002 Elsevier Science Ltd. All rights reserved Keywords: argF gene; arginine biosynthesis; Bacteriodes fragilis; crystal structure; transcarbamylase *Corresponding author Introduction The transcarbamylases are a family of enzymes that catalyze the transfer of a carbamoyl group from carbamoyl phosphate (CP) to an amino or oxygen group of a second substrate. One group of transcarbamylases includes aspartate trans- carbamylase (ATCase), 1 ornithine transcarbamyl- ase (OTCase), 2 oxamate transcarbamylase, 3,4 putrescine transcarbamylase, 5,6 canaline transcarb- amylase 7 and a putative lysine transcarbamylase. 8,9 ATCase and OTCase are the two best studied transcarbamylases. 2,10 The transcarbamylases share a common N-terminal CP-binding domain with a high degree of homology. 11 The C-terminal domains, which bind the second substrate, have more divergent primary sequences but a high degree of conservation at the levels of secondary, tertiary and quaternary structures. 2,12 Little is known about other members of this group, even at the level of primary structure. The second group of transcarbamylases is involved in the biosynthesis of a variety of antibiotics and other metabolites, including venturicidin A, 13 the 3-O-carbamoyl-2-deoxy-b-D-rhamnose moiety of the antifungal antibiotic irumamycin, 14 the related macrolide antibiotic X-149523, 15 and the antitumor antibiotics mitomycins, porfiromycins cephamycin C, 16,17 novobiocin and polyoxins. Among this group, 3-hydroxymethylcephem O-carbamoyl- transferase, which is involved in the synthesis of 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail address of the corresponding author: dshi@childrens-research.org Abbreviations used: ATCase, aspartate transcarbamylase; CP, carbamoyl phosphate; NCS, non- crystallographic symmetry; OTCase, ornithine transcarbamylase; RMS, root mean squared. doi:10.1016/S0022-2836(02)00539-9 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 320, 899–908