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J. Biol. Chem., Vol. 283, Issue 11, 7176-7184, March 14, 2008

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The Crystal Structure of N-Acetyl-L-glutamate Synthase from Neisseria gonorrhoeae Provides Insights into Mechanisms of Catalysis and Regulation^*

Dashuang Shi¹, Vatsala Sagar, Zhongmin Jin^¶, Xiaolin Yu, Ljubica Caldovic, Hiroki Morizono, Norma M. Allewell, and Mendel Tuchman

From the Children's Research Institute, Children's National Medical Center, The George Washington University, Washington, D.C. 20010, Department of Chemistry and Biochemistry, College of Chemical and Life Sciences, University of Maryland, College Park, Maryland 20742, and ^¶Southeast Regional Collaborative Access Team, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439

The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-Å resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-Å linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100Å, respectively, and a height of 110Å_. Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg³¹⁶, Arg⁴²⁵, and Ser⁴²⁷, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys³⁵⁶ and Leu³¹⁴, and hydrophobic interactions to the side chains of Leu³¹³ and Leu³¹⁴. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.

Received for publication, September 12, 2007 , and in revised form, January 7, 2008.

The atomic coordinates and structure factors (code 2R8V, 3B8G, and 2R98) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Public Health Service Grants DK064913 (to M. T.) and DK067935 (to D. S.) from the NIDDK, National Institutes of Health and Grant HD 32652 from the National Institute of Child Health and Human Development. This work was also supported in part by the U. S. Department of Energy under Contract W-31-109-Eng-38. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Children's Research Institute, Children's National Medical Center, 111 Michigan Ave., N.W., Washington, D.C. 20010-2970. Tel.: 202-476-5817; Fax: 202-476-6014; E-mail: dshi{at}cnmcresearch.org.

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