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 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.
- Authors:
-
[6];
[8]
- Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Stanford Univ., CA (United States). Dept. of Chemistry
- Stanford Univ., CA (United States). Dept. of Chemistry
- 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)
- SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
- Stanford Univ., CA (United States). Dept. of Chemistry; SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
- 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)
- 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. https://doi.org/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. https://doi.org/10.1073/pnas.1818274116. https://www.osti.gov/servlets/purl/1528783.
@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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