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Title: Structural Analysis of N-acetylglucosamine-6-phosphate Deacetylase Apoenzyme from Escherichia coli

Abstract

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{sup 2+} 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 Angstroms) iodine anomalous scattering and it was refined against a native dataset up to 2.0 Angstroms 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 ionsmore » 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.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
914031
Report Number(s):
BNL-78599-2007-JA
Journal ID: ISSN 0022-2836; JMOBAK; TRN: US0801487
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Mol. Biol.; Journal Volume: 359; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; 43 PARTICLE ACCELERATORS; BACILLUS SUBTILIS; CHELATING AGENTS; CONFORMATIONAL CHANGES; CRYSTAL STRUCTURE; EMISSION SPECTROSCOPY; ESCHERICHIA COLI; PHOSPHATES; SCATTERING; NSLS; national synchrotron light source

Citation Formats

Ferreira,F., Mendoza-Hernandez, G., Castaneda-Bueno, M., Aparicio, R., Fischer, H., Calcagno, M., and Oliva, G. Structural Analysis of N-acetylglucosamine-6-phosphate Deacetylase Apoenzyme from Escherichia coli. United States: N. p., 2006. Web. doi:10.1016/j.jmb.2006.03.024.
Ferreira,F., Mendoza-Hernandez, G., Castaneda-Bueno, M., Aparicio, R., Fischer, H., Calcagno, M., & Oliva, G. Structural Analysis of N-acetylglucosamine-6-phosphate Deacetylase Apoenzyme from Escherichia coli. United States. doi:10.1016/j.jmb.2006.03.024.
Ferreira,F., Mendoza-Hernandez, G., Castaneda-Bueno, M., Aparicio, R., Fischer, H., Calcagno, M., and Oliva, G. Sun . "Structural Analysis of N-acetylglucosamine-6-phosphate Deacetylase Apoenzyme from Escherichia coli". United States. doi:10.1016/j.jmb.2006.03.024.
@article{osti_914031,
title = {Structural Analysis of N-acetylglucosamine-6-phosphate Deacetylase Apoenzyme from Escherichia coli},
author = {Ferreira,F. and Mendoza-Hernandez, G. and Castaneda-Bueno, M. and Aparicio, R. and Fischer, H. and Calcagno, M. and Oliva, G.},
abstractNote = {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{sup 2+} 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 Angstroms) iodine anomalous scattering and it was refined against a native dataset up to 2.0 Angstroms 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.},
doi = {10.1016/j.jmb.2006.03.024},
journal = {J. Mol. Biol.},
number = 2,
volume = 359,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}