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Proc Natl Acad Sci U S A. 2019 Mar 19;116(12):5370-5375. doi: 10.1073/pnas.1818274116. Epub 2019 Mar 1.

Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
2
Department of Chemistry, Stanford University, Stanford, CA 94305.
3
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
4
Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030.
5
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
6
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; jtainer@mdanderson.org edward.solomon@stanford.edu bertozzi@stanford.edu.
7
Department of Chemistry, Stanford University, Stanford, CA 94305; jtainer@mdanderson.org edward.solomon@stanford.edu bertozzi@stanford.edu.
8
Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305.

Abstract

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.

KEYWORDS:

X-ray spectroscopy; bioinorganic chemistry; copper oxidase; formylglycine; metalloenzyme

PMID:
30824597
DOI:
10.1073/pnas.1818274116

Conflict of interest statement

Conflict of interest statement: C.R.B. is a cofounder and member of the Scientific Advisory Board of Redwood Bioscience (a subsidiary of Catalent, Inc.), which has exclusive rights to the SMARTag technology based on protein modification by FGE. C.R.B. is also a cofounder of Palleon Pharmaceuticals, Enable Biosciences, and InterVenn Biosciences, and a member of the Board of Directors of Eli Lilly & Co.

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