Insights into the mechanism and structure of formylglycine generating enzyme

Miarzlou, Dzmitry. Insights into the mechanism and structure of formylglycine generating enzyme. 2021, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: https://edoc.unibas.ch/84281/

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Formylglycine generating enzyme (FGE) is an essential part of the post-translational machinery that activates sulfatases in pro- and eukaryotes. Any FGE misfunction in humans leads to a multiple sulfatase deficiency (MSD), a fatal lysosomal disorder associated with a drop of the sulfatase activity in vivo. This unique copper-dependent oxidase catalyses the oxygen-dependent conversion of cysteine to formylglycine (fGly), the key residue for sulfatase activity. At the first glance, the FGE active site is unlike any known in mononuclear copper oxidases or oxygenases. The FGE active site is capable of binding and activating O2, although it coordinates the copper in a manner observed in copper-trafficking proteins.
Here, we address the question of a cofactor delivery to the active site prokaryotic FGEs. Analysis of FGE from Thermomonospora curvata (tcFGE) revealed an N-terminal extension (26 aa) as a part of a metal transfer chain, accelerating copper delivery to the active site. The kinetic data suggest that copper-delivery in bacterial systems is a one-way mechanism.
With NMR spectroscopy and protein x-Ray crystallography, we demonstrate that the FGE active site has a Lewis acidic oxygen-binding pocket, juxtaposed to the metal centre. We believe that this key factor allows a tris thiolate copper complex to activate the dioxygen molecule in the FGE active site, in contrast to redox inactive copper-chaperones. Moreover, our first-ever crystal structure of the tertiary complex, E:S:O2, implies the separation of O2-binding and activation as subsequent catalytic steps in the FGE catalytic cycle, unprecedented for mononuclear copper enzymes.
We also show that the conserved tyrosine at the FGE active site possesses conformational flexibility. The structural and kinetic data suggest that this residue is excluded from the catalytic turnover.
At the same time, we use the prokaryotic tcFGE to study a molecular basis of the severe MSD phenotypes caused by the mutations at the Ca binding sites of the Human FGE variant (hsFGE). NMR data, together with Native mass spectrometry, suggests that the two calcium-binding sites, not only significantly contribute to the thermostability and integrity of the FGE fold, but also assure the attomolar copper affinity of the cysteine pair in the FGE active site.
We envision that our findings will not only drive new mechanistic discussions for oxygen activation by tris-thiolate copper complexes but also inspire the design of new synthetic catalysts or artificial enzymes.
Advisors:Seebeck, Florian Peter
Committee Members:Nash, Michael
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Molecular Bionics (Seebeck)
UniBasel Contributors:Seebeck, Florian Peter and Nash, Michael
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14259
Thesis status:Complete
Number of Pages:110
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss142599
edoc DOI:
Last Modified:31 Dec 2023 02:30
Deposited On:09 Sep 2021 08:35

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