Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate
Abstract
Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast—Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PPi. Crystal structures of metal-free Duf89 and Co2+-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl2 and NaF in the presence of divalent cations Co2+, Ni2+, or Zn2+ generated covalent Duf8901-(Asp248)–beryllium trifluoride (BeF3)•Co2+, Duf8901-(Asp248)–BeF3•Ni2+, or Duf8901-(Asp248)–BeF3•Zn2+ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)–phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF3•Co2+ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)–AlF4–OH2•Co2+ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient andmore »
- Authors:
-
[1];
- Memorial Sloan Kettering Cancer Center, New York, NY (United States); Gerstner Sloan Kettering Graduate School of Biomedical Sciences, New York, NY (United States)
- Memorial Sloan Kettering Cancer Center, New York, NY (United States)
- Publication Date:
- Research Org.:
- Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Org.:
- USDOE Office of Science (SC); National Institutes of Health (NIH)
- OSTI Identifier:
- 1866426
- Grant/Contract Number:
- AC02-06CH11357; R35-GM126945; 1746057; P30 GM124165
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Journal of Biological Chemistry
- Additional Journal Information:
- Journal Volume: 298; Journal Issue: 5; Journal ID: ISSN 0021-9258
- Publisher:
- American Society for Biochemistry and Molecular Biology
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; phosphatase; Schizosaccharomyces pombe; protein structure; X-ray crystallography; enzyme mechanism
Citation Formats
Sanchez, Ana M., Jacewicz, Agata, and Shuman, Stewart. Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate. United States: N. p., 2022.
Web. doi:10.1016/j.jbc.2022.101851.
Sanchez, Ana M., Jacewicz, Agata, & Shuman, Stewart. Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate. United States. https://doi.org/10.1016/j.jbc.2022.101851
Sanchez, Ana M., Jacewicz, Agata, and Shuman, Stewart. Fri .
"Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate". United States. https://doi.org/10.1016/j.jbc.2022.101851. https://www.osti.gov/servlets/purl/1866426.
@article{osti_1866426,
title = {Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate},
author = {Sanchez, Ana M. and Jacewicz, Agata and Shuman, Stewart},
abstractNote = {Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast—Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PPi. Crystal structures of metal-free Duf89 and Co2+-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl2 and NaF in the presence of divalent cations Co2+, Ni2+, or Zn2+ generated covalent Duf8901-(Asp248)–beryllium trifluoride (BeF3)•Co2+, Duf8901-(Asp248)–BeF3•Ni2+, or Duf8901-(Asp248)–BeF3•Zn2+ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)–phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF3•Co2+ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)–AlF4–OH2•Co2+ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.},
doi = {10.1016/j.jbc.2022.101851},
journal = {Journal of Biological Chemistry},
number = 5,
volume = 298,
place = {United States},
year = {Fri Mar 18 00:00:00 EDT 2022},
month = {Fri Mar 18 00:00:00 EDT 2022}
}
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