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High-Resolution Analysis of Zn^2+ Coordination in the Alkaline Phosphatase Superfamily by EXAFS and X-ray Crystallography

Bobyr, Elena and Lassila, Jonathan K. and Wiersma-Koch, Helen I. and Fenn, Timothy D. and Lee, Jason J. and Nikolic-Hughes, Ivana and Hodgson, Keith O. and Rees, Douglas C. and Hedman, Britt and Herschlag, Daniel (2012) High-Resolution Analysis of Zn^2+ Coordination in the Alkaline Phosphatase Superfamily by EXAFS and X-ray Crystallography. Journal of Molecular Biology, 415 (1). pp. 102-117. ISSN 0022-2836. PMCID PMC3249517. doi:10.1016/j.jmb.2011.10.040. https://resolver.caltech.edu/CaltechAUTHORS:20120227-122308180

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Abstract

Comparisons among evolutionarily related enzymes offer opportunities to reveal how structural differences produce different catalytic activities. Two structurally related enzymes, Escherichia coli alkaline phosphatase (AP) and Xanthomonas axonopodis nucleotide pyrophosphatase/phosphodiesterase (NPP), have nearly identical binuclear Zn^2+ catalytic centers but show tremendous differential specificity for hydrolysis of phosphate monoesters or phosphate diesters. To determine if there are differences in Zn^2+ coordination in the two enzymes that might contribute to catalytic specificity, we analyzed both x-ray absorption spectroscopic and x-ray crystallographic data. We report a 1.29-Å crystal structure of AP with bound phosphate, allowing evaluation of interactions at the AP metal site with high resolution. To make systematic comparisons between AP and NPP, we measured zinc extended x-ray absorption fine structure for AP and NPP in the free-enzyme forms, with AMP and inorganic phosphate groundstate analogs and with vanadate transition-state analogs. These studies yielded average zinc–ligand distances in AP and NPP free-enzyme forms and ground-state analog forms that were identical within error, suggesting little difference in metal ion coordination among these forms. Upon binding of vanadate to both enzymes, small increases in average metal–ligand distances were observed, consistent with an increased coordination number. Slightly longer increases were observed in NPP relative to AP, which could arise from subtle rearrangements of the active site or differences in the geometry of the bound vanadyl species. Overall, the results suggest that the binuclear Zn^2+ catalytic site remains very similar between AP and NPP during the course of a reaction cycle.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.jmb.2011.10.040DOIArticle
http://www.sciencedirect.com/science/article/pii/S0022283611011776PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249517/PubMed CentralArticle
ORCID:
AuthorORCID
Rees, Douglas C.0000-0003-4073-1185
Additional Information:© 2011 Elsevier Ltd. Received 17 August 2011; received in revised form 21 October 2011; accepted 24 October 2011. Available online 28 October 2011. We thank members of the Herschlag laboratory for helpful comments on the manuscript, Dr. Jun-yong Choe for assistance with diffraction data collection, Dr. Stefan Steinbacher for participating in the initial crystallographic refinement, Dr. Axel T. Brunger for the use of facilities for crystallographic refinement, and Jesse G. Zalatan for sharing unpublished NPP binding data. This work was supported by grants from the National Institutes of Health (NIH) to D.H. (GM64798), D.C.R., (GM045162), and K.O.H. (RR001209). J.K.L. was supported by an NIH postdoctoral fellowship (F32 GM080865). T.D.F. was supported by the Universitywide AIDS Research Program of the University of California (F03-ST-216). Facilities used for x-ray crystallography in the laboratory of Axel T. Brunger were supported by Howard Hughes Medical Institute. Portions of this research were carried out at the SSRL, a Directorate of Stanford Linear Accelerator Center National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the NIH, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences. The publication was partially supported by grant number 5 P41 RR001209 from the National Center for Research Resources, a component of the NIH, and its contents are solely the responsibility of the authors and do not necessarily represent the official view of the National Center for Research Resources or the NIH.
Funders:
Funding AgencyGrant Number
NIHGM64798
NIHGM045162
NIHRR001209
NIH postdoctoral fellowshipF32 GM080865
University of California Universitywide AIDS Research ProgramF03-ST-216
NIH5 P41 RR001209
Subject Keywords:x-ray absorption spectroscopy; crystal structure; nucleotide pyrophosphatase/phosphodiesterase; catalytic promiscuity; phosphoryl transfer
Issue or Number:1
PubMed Central ID:PMC3249517
DOI:10.1016/j.jmb.2011.10.040
Record Number:CaltechAUTHORS:20120227-122308180
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20120227-122308180
Official Citation:Elena Bobyr, Jonathan K. Lassila, Helen I. Wiersma-Koch, Timothy D. Fenn, Jason J. Lee, Ivana Nikolic-Hughes, Keith O. Hodgson, Douglas C. Rees, Britt Hedman, Daniel Herschlag, High-Resolution Analysis of Zn2+ Coordination in the Alkaline Phosphatase Superfamily by EXAFS and X-ray Crystallography, Journal of Molecular Biology, Volume 415, Issue 1, 6 January 2012, Pages 102-117, ISSN 0022-2836, 10.1016/j.jmb.2011.10.040.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:29486
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:28 Feb 2012 21:16
Last Modified:09 Nov 2021 17:07

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