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Title: Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

Abstract

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration ofmore » enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1005703
Resource Type:
Journal Article
Journal Name:
Biochemistry-US
Additional Journal Information:
Journal Volume: 48; Journal Issue: (2) ; 01, 2009; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
ENGLISH
Subject:
08 HYDROGEN; 36 MATERIALS SCIENCE; ATP; BACTERIA; CARBOXYLIC ACIDS; CATABOLISM; CONFORMATIONAL CHANGES; CRYSTAL STRUCTURE; DIMERS; ENZYMES; GLYCEROL; HISTIDINE; HYDROGEN; INTERACTIONS; INTERFACES; METABOLISM; MUTANTS; MUTATIONS; PHOSPHORYLATION; PHOSPHOTRANSFERASES; POSITIONING; PRODUCTION; RESIDUES; SACCHAROSE; SIGNALS; STABILITY; SUBSTRATES; UPTAKE

Citation Formats

Yeh, Joanne I, Kettering, Regina, Saxl, Ruth, Bourand, Alexa, Darbon, Emmanuelle, Joly, Nathalie, Briozzo, Pierre, Deutscher, Josef, Pitt), and CNRS-CRMD). Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation. United States: N. p., 2009. Web. doi:10.1021/bi8009407.
Yeh, Joanne I, Kettering, Regina, Saxl, Ruth, Bourand, Alexa, Darbon, Emmanuelle, Joly, Nathalie, Briozzo, Pierre, Deutscher, Josef, Pitt), & CNRS-CRMD). Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation. United States. https://doi.org/10.1021/bi8009407
Yeh, Joanne I, Kettering, Regina, Saxl, Ruth, Bourand, Alexa, Darbon, Emmanuelle, Joly, Nathalie, Briozzo, Pierre, Deutscher, Josef, Pitt), and CNRS-CRMD). 2009. "Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation". United States. https://doi.org/10.1021/bi8009407.
@article{osti_1005703,
title = {Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation},
author = {Yeh, Joanne I and Kettering, Regina and Saxl, Ruth and Bourand, Alexa and Darbon, Emmanuelle and Joly, Nathalie and Briozzo, Pierre and Deutscher, Josef and Pitt) and CNRS-CRMD)},
abstractNote = {Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.},
doi = {10.1021/bi8009407},
url = {https://www.osti.gov/biblio/1005703}, journal = {Biochemistry-US},
issn = {0006-2960},
number = (2) ; 01, 2009,
volume = 48,
place = {United States},
year = {Fri Sep 11 00:00:00 EDT 2009},
month = {Fri Sep 11 00:00:00 EDT 2009}
}