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Crystal Structure of a HyperactiveEscherichia coliGlycerol Kinase Mutant Gly230 → Asp Obtained Using Microfluidic Crystallization Devices

Anderson, Megan J. and DeLaBarre, Byron and Raghunathan, Anu and Palsson, Bernhard O. and Brunger, Axel T. and Quake, Stephen R. (2007) Crystal Structure of a HyperactiveEscherichia coliGlycerol Kinase Mutant Gly230 → Asp Obtained Using Microfluidic Crystallization Devices. Biochemistry, 46 (19). pp. 5722-5731. ISSN 0006-2960. doi:10.1021/bi700096p. https://resolver.caltech.edu/CaltechAUTHORS:20170201-074621783

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Abstract

The crystal structure of an Escherichia coli glycerol kinase mutant Gly230 → Asp (GKG230D) was determined to 2.0 Å resolution using a microfluidics based crystallization platform. The crystallization strategy involved a suite of microfluidic devices that characterized the solubility trends of GKG230D, performed nanoliter volume free interface diffusion crystallization experiments, and produced diffraction-quality crystals for in situ data collection. GKG230D displays increased enzymatic activity and decreased allosteric regulation by the glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared to wild-type GK (GKWT). Structural analysis revealed that the decreased allosteric regulation is a result of the altered FBP binding loop conformations in GKG230D that interfere with the wild-type FBP binding site. The altered FBP binding loop conformations in GKG230D are supported through a series of intramolecular loop interactions. The appearance of Asp230 in the FBP binding loops also repositions the wild-type FBP binding residues away from the FBP binding site. Light scattering analysis confirmed GKG230D is a dimer and is resistant to tetramer formation in the presence of FBP, whereas GKWT dimers are converted into putatively inactive tetramers in the presence of FBP. GKG230D also provides the first structural evidence for multiple GK monomer conformations in the presence of glycerol and in the absence of a nucleotide substrate and verifies that glycerol binding is not responsible for locking GK into the closed conformation necessary for GK activity.


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http://dx.doi.org/10.1021/bi700096pDOIArticle
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Additional Information:© 2007 American Chemical Society. Received 17 January 2007. Published online 19 April 2007. Published in print 1 May 2007. This work was supported in part by the Howard Hughes Medical Institute and the NIH Director’s Pioneer Award to S.R.Q. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences.
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Howard Hughes Medical Institute (HHMI)UNSPECIFIED
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Department of Energy (DOE)UNSPECIFIED
National Institute of General Medical SciencesUNSPECIFIED
Issue or Number:19
DOI:10.1021/bi700096p
Record Number:CaltechAUTHORS:20170201-074621783
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170201-074621783
Official Citation:Crystal Structure of a Hyperactive Escherichia coli Glycerol Kinase Mutant Gly230 → Asp Obtained Using Microfluidic Crystallization Devices Megan J. Anderson, Byron DeLaBarre, Anu Raghunathan, Bernhard O. Palsson, Axel T. Brunger, and Stephen R. Quake Biochemistry 2007 46 (19), 5722-5731 DOI: 10.1021/bi700096p
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73903
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:01 Feb 2017 17:07
Last Modified:11 Nov 2021 05:23

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