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Title: Structure of a Class I Tagatose-1,6-bisphosphate Aldolase - Investigation into an Apparent Loss of Stereospecificity

Abstract

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a ({alpha}/{beta}){sub 8} fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys{sup 205}, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R andmore » pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2-C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase.« less

Authors:
; ;
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
Sponsoring Org.:
DOE - OFFICE OF SCIENCE
OSTI Identifier:
1019666
Report Number(s):
BNL-95512-2011-JA
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US201115%%306
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 285; Journal Issue: 27; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
English
Subject:
08 HYDROGEN; ALDOLASES; ANIONS; CARBON; CLEAVAGE; CONFIGURATION; CRYSTAL STRUCTURE; CRYSTALLOGRAPHY; ELECTRON DENSITY; ENZYMES; FRUCTOSE; HYDROGEN; KINETICS; PHOSPHATES; PROTEIN STRUCTURE; PROTONS; RESOLUTION; SORBOSE; SPECTROSCOPY; STREPTOCOCCUS; TRITIUM OXIDES; national synchrotron light source

Citation Formats

LowKam, C, Liotard, B, and Sygusch, J. Structure of a Class I Tagatose-1,6-bisphosphate Aldolase - Investigation into an Apparent Loss of Stereospecificity. United States: N. p., 2010. Web. doi:10.1074/jbc.M109.080358.
LowKam, C, Liotard, B, & Sygusch, J. Structure of a Class I Tagatose-1,6-bisphosphate Aldolase - Investigation into an Apparent Loss of Stereospecificity. United States. https://doi.org/10.1074/jbc.M109.080358
LowKam, C, Liotard, B, and Sygusch, J. 2010. "Structure of a Class I Tagatose-1,6-bisphosphate Aldolase - Investigation into an Apparent Loss of Stereospecificity". United States. https://doi.org/10.1074/jbc.M109.080358.
@article{osti_1019666,
title = {Structure of a Class I Tagatose-1,6-bisphosphate Aldolase - Investigation into an Apparent Loss of Stereospecificity},
author = {LowKam, C and Liotard, B and Sygusch, J},
abstractNote = {Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a ({alpha}/{beta}){sub 8} fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys{sup 205}, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2-C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase.},
doi = {10.1074/jbc.M109.080358},
url = {https://www.osti.gov/biblio/1019666}, journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 27,
volume = 285,
place = {United States},
year = {Fri Jan 01 00:00:00 EST 2010},
month = {Fri Jan 01 00:00:00 EST 2010}
}