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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}
}
  • The peptidoglycan N-acetylglucosamine (GlcNAc) deacetylase BC1960 from B. cereus was crystallized in the presence of the substrate (GlcNAc){sub 6}. The crystals belonged to space group P4{sub 1}2{sub 1}2 and diffracted to 2.38 Å resolution. The peptidoglycan N-acetylglucosamine (GlcNAc) deacetylase BC1960 from Bacillus cereus (EC 3.5.1.33), an enzyme consisting of 275 amino acids, was crystallized in the presence of its substrate (GlcNAc){sub 6}. The crystals belonged to the tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 92.7, c = 242.9 Å and four molecules in the asymmetric unit. A complete data set was collected at 100more » K to a resolution of 2.38 Å using synchrotron radiation.« less
  • N-Acetylglucosamine (GlcNAc) is generated, in liver and other tissues, as a product of the lysosomal degradation of complex carbohydrates. After diffusion into the cytosol, the monosaccharide is metabolized via catabolic or anabolic pathways, in which the first step is common to both pathways and consists of phosphorylation at C-6 by a specific GlcNAc kinase. When processed via the catabolic pathway, GlcNAc 6-phosphate is then deacetylated to glucosamine (GlcN) 6-phosphate. In the present study, they have developed a simple assay for the deacetylase catalyzing this reaction, in which /sup 3/H-acetate released from /sup 3/H-acetyl-labeled GlcNAc 6-phosphate is measured directly in amore » biphasic liquid scintillation counting system after acidification of the reaction mixture. The new assay has been used in partial purification of the deacetylase from rat liver and in analysis of kinetic and other properties of the enzyme. GlcN 6-phosphate and glucose 6-phosphate inhibited the deacetylase, while GlcNAc, GlcNAc 1-phosphate, GlcN 1-phosphate, and N-acetylgalactosamine had no effect. The activity of the enzyme was substantially inhibited by 5 mM Mn/sup 2 +/, Ni/sup 2 +/, or Cu/sup 2 +/, while Ca/sup 2 +/, Mg/sup 2 +/, and Ba/sup 2 +/ had essentially no effect at concentrations of 12.5 mM or lower. Co/sup 2 +/ stimulated at low concentrations (< 5 mM) but inhibited at higher concentrations. Gel chromatography on Sepharose CL-6B indicated that the enzyme had a mol. wt. greater than 160,000.« less
  • Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies ofmore » d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate group hydrogen bonds not only with the conserved motif but also with an active site loop following the sixth {beta}-strand, providing a potential structural mechanism for coupling substrate binding with catalysis.« less
  • N-(5'-Phosphoribosyl) anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli is a monomeric bifunctional enzyme of M/sub r/ 49,500 that catalyzes two sequential reactions in the biosynthesis of tryptophan. The three-dimensional structure of the enzyme has been determined at 2.8-A resolution by x-ray crystallography. The two catalytic activities reside on distinct functional domains of similar folding, that of an eightfold parallel ..beta..-barrel with ..cap alpha..-helices on the outside connecting the ..beta..-strands. Both active sites were located with an iodinated substrate analogue and found to be in depressions on the surface of the domains created by the outward-curving hoops between the carboxyl termini ofmore » the ..beta..-sheet strands and the subsequent ..cap alpha..-helices. They do not face each other, making channeling of the substrate between active sties virtually impossible. Despite the structural similarity of the two domains, no significant sequence homology was found when topologically equivalent residues were compared« less