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A family of metal-dependent phosphatases implicated in metabolite damage-control

Nature Chemical Biology volume 12, pages 621–627 (2016)Cite this article

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Abstract

DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed a novel phosphatase active site with fructose 6-phosphate and Mg2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.

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Figure 1: The three subfamilies of DUF89 proteins.
Figure 2: Substrate profiles of DUF89 proteins from each subfamily.
Figure 3: Phosphatase activities of pantothenate kinase DUF89 domains in relation to CoA synthesis and salvage.
Figure 4: Hexose metabolism in wild-type and ΔYMR027W yeast strains.
Figure 5: Overall structures of DUF89 protomers.
Figure 6: Close-up views of the active site of DUF89 proteins.

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Acknowledgements

This work was supported by US National Science Foundation grants MCB-1153413 and IOS-1025398 (to A.D.H.) and MCB-1153491 (to O.F.); by Genome Canada, the Ontario Genomics Institute (2009-OGI-ABC-1405), the Ontario Research Fund (ORF-GL2-01-004), and NSERC Strategic Network grant IBN (to A.F.Y.); by National Institutes of Health grant GM094585 and the US Department of Energy, Office of Biological and Environmental Research, contract DE-AC02-06CH11357 (to A.J.); and by the C.V. Griffin Sr. Foundation (to A.D.H.). J.S. was supported by US Department of Energy, Office of Basic Energy Sciences, grant DOE KC0304000. We thank J.E. Cronan and H.W. Park for enzymes, K. Kodama of Ikeda Corp. of America for pantetheine-S-sulfonate, S. Gerdes for bioinformatic help, E. van Schaftingen for advice on metabolism, and J.D. Rabinowitz for pilot metabolomics.

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  1. Lili Huang and Anna Khusnutdinova: These authors contributed equally to this work.

Authors and Affiliations

  1. Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida, USA

    Lili Huang & Jesse F Gregory III

  2. Department of Chemical Engineering & Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

    Anna Khusnutdinova, Greg Brown, Xiaohui Xu, Hong Cui, Pierre Petit, Robert Flick, Alexei Savchenko & Alexander F Yakunin

  3. Biosciences Division, Midwest Center for Structural Genomics and Structural Biology Center, Argonne National Laboratory, Argonne, Illinois, USA

    Boguslaw Nocek & Andrzej Joachimiak

  4. Microbiology and Cell Science Department, University of Florida, Gainesville, Florida, USA

    Rémi Zallot & Valérie de Crécy-Lagard

  5. Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida, USA

    Kelly Balmant

  6. Horticultural Sciences Department, University of Florida, Gainesville, Florida, USA

    Michael J Ziemak & Andrew D Hanson

  7. and Climate Sciences Department, Biological, Environmental, Brookhaven National Laboratory, Upton, New York, USA

    John Shanklin

  8. Metabolomics Core, UC Davis Genome Center, University of California, Davis, Davis, California, USA.,

    Oliver Fiehn

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  1. Lili Huang
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Contributions

A.D.H. and A.F.Y. conceived the project. K.B., R.Z., V.d.C.-L., and A.D.H. made comparative genomic analyses. L.H., A.K., G.B., R.F., M.J.Z., J.S., and J.F.G. carried out biochemical experiments. O.F. performed metabolomics analyses. H.C., A.J., B.N., P.P., A.S., and X.X. performed crystallographic studies. A.D.H., L.H., and A.F.Y. wrote the manuscript.

Corresponding authors

Correspondence to Alexander F Yakunin or Andrew D Hanson.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–7 and Supplementary Figures 1–11. (PDF 1938 kb)

Supplementary Data Set 1

Untargeted metabolomics analysis of yeast wild type and YMR027W deletion strains. (XLSX 333 kb)

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Huang, L., Khusnutdinova, A., Nocek, B. et al. A family of metal-dependent phosphatases implicated in metabolite damage-control. Nat Chem Biol 12, 621–627 (2016). https://doi.org/10.1038/nchembio.2108

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