Structural Analysis of N-acetylglucosamine- 6-phosphate Deacetylase Apoenzyme from Escherichia coli Frederico M. Ferreira 1 , Guillermo Mendoza-Hernandez 2 Maria Castan ˜ eda-Bueno 2 , Ricardo Aparicio 3 , Hannes Fischer 1 Mario L. Calcagno 2 and Glaucius Oliva 1 * 1 Instituto de Fı ´sica de Sa ˜o Carlos, Universidade de Sa ˜o Paulo, C.P. 369, 13560-970 Sa ˜o Carlos, SP, Brazil 2 Departamento de Bioquı ´mica Facultad de Medicina Universidad Nacional Auto ´noma de Mexico AP. Postal 70-159 Cd. Universitaria, 04510 Mexico, DF Mexico 3 Instituto de Quı ´mica Universidade Estadual de Campinas, C.P. 6154, 13084-862 Campinas, SP, Brazil We report the crystal structure of the apoenzyme of N-acetylglucosamine- 6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn 2C in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A ˚ ) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A ˚ resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer–dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis. q 2006 Elsevier Ltd. All rights reserved. Keywords: N-acetylglucosamine-6-phosphate deacetylase; amino sugar catabolism; amidohydrolases; PIXE; ICP-AES *Corresponding author Introduction The bacterium Escherichia coli displays a great versatility in the utilization of different carbon sources and it can use a great variety of carbohydrates and related compounds as sources of energy. When amino sugars, glucosamine (GlcN) or N-acetylglucosamine (GlcNAc), are present in the environment, they are taken up by the cells and used for cell wall and lipid A synthesis as well as a carbon source. Under these conditions, the synthesis of the enzyme glucosamine synthase (GlmS) necessary for the biosynthesis of amino sugars, is repressed while the enzymes necessary for the transport and meta- bolism of amino sugars are induced. 1,2 GlcNAc is amongst the best carbon sources known for E. coli, producing similar growth rates as glucose. In E. coli both GlcN and GlcNAc are phosphotransferase 0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. Abbreviations used: GlcNAc6P, N-acetylglucosamine 6-phosphate; NAGPase, N-acetylglucosamine- 6-phosphate deacetylase; SIRAS, single isomorphous replacement with anomalous scattering; SAXS, small- angle X-ray scattering; PIXE, photon-induced X-ray emission; ICP-AES, inductively coupled plasma emission spectroscopy; DRM, dummy residues model. E-mail address of the corresponding author: oliva@if.sc.usp.br doi:10.1016/j.jmb.2006.03.024 J. Mol. Biol. (2006) 359, 308–321