Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli.
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 Zn2+ 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.
Ferreira
Frederico M
FM
Instituto de Física de São Carlos, Universidade de São Paulo, C.P. 369, 13560-970 São Carlos, SP, Brazil.
Mendoza-Hernandez
Guillermo
G
Castañeda-Bueno
Maria
M
Aparicio
Ricardo
R
Fischer
Hannes
H
Calcagno
Mario L
ML
Oliva
Glaucius
G
D000581:Amidohydrolases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020124:Thermotoga maritima / Q000201:enzymology; D015032:Zinc / Q000737:chemistry
D029968:Escherichia coli Proteins; D008667:Metalloproteins; D000581:Amidohydrolases; C018717:N-acetylglucosamine-6-phosphate deacetylase; D015032:Zinc