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Published January 6, 2012 | public
Journal Article Open

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


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.

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.

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August 22, 2023
August 22, 2023