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Title: Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O 2 activation

Abstract

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to C α-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 O 2activation 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 O 2 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.

Authors:
 [1];  [2];  [3];  [2];  [4];  [5];  [6];  [7]; ORCiD logo [6]; ORCiD logo [8]
  1. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Stanford Univ., CA (United States). Dept. of Chemistry
  2. Stanford Univ., CA (United States). Dept. of Chemistry
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division
  4. Univ. of Texas MD Anderson Cancer Center, Houston, TX (United States)
  5. SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
  6. Stanford Univ., CA (United States). Dept. of Chemistry; SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
  7. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Univ. of Texas MD Anderson Cancer Center, Houston, TX (United States)
  8. Stanford Univ., CA (United States). Dept. of Chemistry, and Howard Hughes Medical Inst.
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE; National Institutes of Health (NIH)
OSTI Identifier:
1528783
Grant/Contract Number:  
AC02-76SF00515; R01DK031450; F32GM116240; R35CA22043; CA227942
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 116; Journal Issue: 12; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Appel, Mason J., Meier, Katlyn K., Lafrance-Vanasse, Julien, Lim, Hyeongtaek, Tsai, Chi-Lin, Hedman, Britt, Hodgson, Keith O., Tainer, John A., Solomon, Edward I., and Bertozzi, Carolyn R. Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O 2 activation. United States: N. p., 2019. Web. doi:10.1073/pnas.1818274116.
Appel, Mason J., Meier, Katlyn K., Lafrance-Vanasse, Julien, Lim, Hyeongtaek, Tsai, Chi-Lin, Hedman, Britt, Hodgson, Keith O., Tainer, John A., Solomon, Edward I., & Bertozzi, Carolyn R. Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O 2 activation. United States. doi:10.1073/pnas.1818274116.
Appel, Mason J., Meier, Katlyn K., Lafrance-Vanasse, Julien, Lim, Hyeongtaek, Tsai, Chi-Lin, Hedman, Britt, Hodgson, Keith O., Tainer, John A., Solomon, Edward I., and Bertozzi, Carolyn R. Fri . "Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O 2 activation". United States. doi:10.1073/pnas.1818274116.
@article{osti_1528783,
title = {Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O 2 activation},
author = {Appel, Mason J. and Meier, Katlyn K. and Lafrance-Vanasse, Julien and Lim, Hyeongtaek and Tsai, Chi-Lin and Hedman, Britt and Hodgson, Keith O. and Tainer, John A. and Solomon, Edward I. and Bertozzi, Carolyn R.},
abstractNote = {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 O2activation 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.},
doi = {10.1073/pnas.1818274116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 12,
volume = 116,
place = {United States},
year = {2019},
month = {3}
}

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