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Title: Crystal Structure of Mn2+-bound Escherichia coli L-arabinose Isomerase (ECAI) and Implications in Protein Catalytic Mechanism and Thermo-Stability

Abstract

The functional properties of proteins depend on their three-dimensional shapes. Protein structures can be determined by X-ray crystallography as a tool. The three-dimensional structure of the apo form of the Escherichia coli L-arabinose isomerase (ECAI) has recently been determined. ECAI is responsible for the initial stage of L-arabinose catabolism, converting arabinose into ribulose in vivo. This enzyme also plays a crucial role in catalyzing the conversion of galactose into tagatose (low calorie natural sugar) in vitro. ECAI utilizes Mn{sup 2+} for its catalytic activity. Crystals of the ECAI + Mn{sup 2+} complex helps to investigate the catalytic properties of the enzyme. Therefore, crystals of ECAI + Mn{sup 2+} complex were grown using hanging drop vapor diffusion method at room temperature. Diffraction data were collected at X4C beamline, National Synchrotron Light Source, Brookhaven National Laboratory. The structure was solved by the molecular replacement technique and has been refined to Rwork of 0.23 at 2.8 {angstrom} resolution using X3A beamline computational facility. The structure was deposited to Protein Data Bank (PDB ID 2HXG). Mn{sup 2+} ion was localized to the previously identified putative active site with octahedral coordination. Comparison of apo and holo form of ECAI structures permits the identification of structuralmore » features that are of importance to the intrinsic activity and heat stability of AI.« less

Authors:
; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930523
Report Number(s):
BNL-80530-2008-JA
Journal ID: ISSN 1470-8175; TRN: US0901406
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Young Investigators; Journal Volume: 17; Journal Issue: 3
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; 43 PARTICLE ACCELERATORS; ARABINOSE; BNL; CATABOLISM; CRYSTAL STRUCTURE; CRYSTALLOGRAPHY; DIFFRACTION; DIFFUSION; ENZYMES; ESCHERICHIA COLI; FUNCTIONALS; GALACTOSE; IN VITRO; IN VIVO; ISOMERASES; NSLS; PROTEIN STRUCTURE; PROTEINS; RESOLUTION; RIBULOSE; SACCHAROSE; SENSITIVITY; THERMAL DEGRADATION; national synchrotron light source

Citation Formats

Zhu,W., Manjasetty, B., and Chance, M. Crystal Structure of Mn2+-bound Escherichia coli L-arabinose Isomerase (ECAI) and Implications in Protein Catalytic Mechanism and Thermo-Stability. United States: N. p., 2007. Web.
Zhu,W., Manjasetty, B., & Chance, M. Crystal Structure of Mn2+-bound Escherichia coli L-arabinose Isomerase (ECAI) and Implications in Protein Catalytic Mechanism and Thermo-Stability. United States.
Zhu,W., Manjasetty, B., and Chance, M. Mon . "Crystal Structure of Mn2+-bound Escherichia coli L-arabinose Isomerase (ECAI) and Implications in Protein Catalytic Mechanism and Thermo-Stability". United States. doi:.
@article{osti_930523,
title = {Crystal Structure of Mn2+-bound Escherichia coli L-arabinose Isomerase (ECAI) and Implications in Protein Catalytic Mechanism and Thermo-Stability},
author = {Zhu,W. and Manjasetty, B. and Chance, M.},
abstractNote = {The functional properties of proteins depend on their three-dimensional shapes. Protein structures can be determined by X-ray crystallography as a tool. The three-dimensional structure of the apo form of the Escherichia coli L-arabinose isomerase (ECAI) has recently been determined. ECAI is responsible for the initial stage of L-arabinose catabolism, converting arabinose into ribulose in vivo. This enzyme also plays a crucial role in catalyzing the conversion of galactose into tagatose (low calorie natural sugar) in vitro. ECAI utilizes Mn{sup 2+} for its catalytic activity. Crystals of the ECAI + Mn{sup 2+} complex helps to investigate the catalytic properties of the enzyme. Therefore, crystals of ECAI + Mn{sup 2+} complex were grown using hanging drop vapor diffusion method at room temperature. Diffraction data were collected at X4C beamline, National Synchrotron Light Source, Brookhaven National Laboratory. The structure was solved by the molecular replacement technique and has been refined to Rwork of 0.23 at 2.8 {angstrom} resolution using X3A beamline computational facility. The structure was deposited to Protein Data Bank (PDB ID 2HXG). Mn{sup 2+} ion was localized to the previously identified putative active site with octahedral coordination. Comparison of apo and holo form of ECAI structures permits the identification of structural features that are of importance to the intrinsic activity and heat stability of AI.},
doi = {},
journal = {Journal of Young Investigators},
number = 3,
volume = 17,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • Proteins are comprised of one or multiple versions of long chains of connected amino acids known as polypeptides. The human body alone has been estimated to have approximately two million proteins, and of all land based biological organisms, it is predicted that tens of millions of different proteins exist. Despite this, however, only about one million proteins have been documented to date, of which barely a fraction has been thoroughly analyzed. It is for this reason that the quality research performed by Cornell undergraduate student Weisha Zhu, and her advisors Babu Manjasetty and Mark R. Chance, is of such greatmore » importance.« less
  • Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 Angstroms resolution. The subunit structure of ECAI is organized into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexamericmore » in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.« less
  • Genetic and enzymatic analysis of 17 L-arabinose nonutilizing mutants of Escherichia coli B/r, together with eight previously analyzed mutants of the B gene, show no relationship between the location of a mutant site in the B gene and its effect on the level of inducib1e L-arabinose isomerase, the product of the adjacent structural A gene. The results of reciprocal three-factor crosses were used to estab1ish the linear genetic order of most arabinose negative mutants. Other mutants could not be ordered by this method because of high negative interference or close linkage. Recombination distances were used to establish the probable ordermore » of most of these mutants. (auth)« less
  • Gamma irradiation of Escherichia coli B/r caused a dose-dependent inhibition of the capacity of the cells to synthesize L-arabinose isomerase in response to the inducer. At higher doses (18 krad and above), postirradiation incubation led to further inhibition of the capacity to synthesize L-arabinose isomerase, whereas cells receiving lower doses recovered from the damage to the enzyme synthesizing system following incubation. Cyclic AMP partially reversed the inhibitory effect on L-arabinose isomerase induction produced immediately after irradiation by all gamma-ray doses (up to 30 krad), but the enhanced inhibitory effect caused by induction in cells irradiated at higher doses could notmore » be reversed by the nucleotide. It is suggested that although catabolite repression is partly responsible for causing the inhibition of the enzyme synthesizing capacity of the cells observed immediately after gamma irradiation, the enhanced inhibition caused by incubating cells irradiated at higher doses is not due to interference with the control mechanism regulated by catabolite repression.« less