Formylglycine-Generating Enzyme Binds Substrate Directly at a Mononuclear Cu(I) Center to Initiate O2 Activation Mason J

Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation Mason J. Appela,b,1, Katlyn K. Meierb,1, Julien Lafrance-Vanassec,2, Hyeongtaek Limb, Chi-Lin Tsaid, Britt Hedmane, Keith O. Hodgsonb,e, John A. Tainerc,d,3, Edward I. Solomonb,e,3, and Carolyn R. Bertozzib,f,3 aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; bDepartment of Chemistry, Stanford University, Stanford, CA 94305; cMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; dDepartment of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; eStanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025; and fHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305 Edited by James E. Penner-Hahn, University of Michigan, Ann Arbor, MI, and accepted by Editorial Board Member Stephen J. Benkovic January 29, 2019 (received for review October 23, 2018) The formylglycine-generating enzyme (FGE) is required for the Likely owing to the difficulty of isolating metallated Cu(I) protein posttranslational activation of type I sulfatases by oxidation of an crystals (13), structural characterization of an authentic Cu–FGE active-site cysteine to Cα-formylglycine. FGE has emerged as an complex has remained elusive. Additionally, the manner in which enabling biotechnology tool due to the robust utility of the alde- the monocopper site reacts with O2, and the reactive intermedi- hyde product as a bioconjugation handle in recombinant proteins. ates responsible for substrate oxidation are unclear. Therefore, Here, we show that Cu(I)–FGE is functional in O2 activation and elucidation of the catalytic mechanism of FGE is fundamental to a reveal a high-resolution X-ray crystal structure of FGE in complex broader understanding of copper-mediated biotransformations. with its catalytic copper cofactor. We establish that the copper Here, we present the X-ray crystal structure of Cu-bound FGE atom is coordinated by two active-site cysteine residues in a nearly at a resolution of 2.2 Å. The linear cuprous dithiolate ligand linear geometry, supporting and extending prior biochemical and system observed in the holoenzyme structure is unusual for a CHEMISTRY structural data. The active cuprous FGE complex was interrogated copper oxidase and is superficially similar to well-characterized directly by X-ray absorption spectroscopy. These data unambigu- Cu(I) transporters and chaperones (14). Additionally, we report ously establish the configuration of the resting enzyme metal cen- the electronic absorption and electron paramagnetic resonance ter and, importantly, reveal the formation of a three-coordinate (EPR) features of a transient cupric-FGE complex, providing tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calcula- Significance tions. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a Many enzymes harness the energy of molecular oxygen to cata- BIOCHEMISTRY detailed mechanistic framework for understanding why nature lyze diverse transformations. However, reaction with oxygen is chose this structurally unique monocopper active site to catalyze kinetically challenging and must be precisely controlled to prevent oxidase chemistry for sulfatase activation. cellular damage. The formylglycine-generating enzyme activates its targets by utilizing a mononuclear copper center to facilitate formylglycine | copper oxidase | metalloenzyme | X-ray spectroscopy | oxidation of a substrate cysteine. We reveal the structure of its bioinorganic chemistry atypical copper active site and discover that substrate binding directly to copper precedes and initiates reactivity toward oxygen. he formylglycine-generating enzyme (FGE) catalyzes the We furthermore identify a transient intermediate that may pro- Tcotranslational or posttranslational activation of type I sul- vide evidence for the involvement of an elusive catalytic species. fatases in eukaryotes and aerobic microbes (1). This is accom- This work uncovers a distinct strategy among copper enzymes plished by oxidation of a sulfatase active-site cysteine residue that exploits a dynamic binding site to tightly couple the activa- located in the minimum consensus sequence CXPXR to Cα- tion of oxygen with selective substrate oxidation. formylglycine (fGly), a critical cofactor for the hydrolysis of sulfate ester substrates (Fig. 1). Dysfunction of FGE in humans Author contributions: M.J.A., K.K.M., J.L.-V., J.A.T., E.I.S., and C.R.B. designed research; results in a congenital disease, multiple sulfatase deficiency, M.J.A., K.K.M., J.L.-V., and H.L. performed research; M.J.A., K.K.M., J.L.-V., H.L., C.-L.T., B.H., K.O.H., J.A.T., E.I.S., and C.R.B. analyzed data; and M.J.A., K.K.M., J.A.T., E.I.S., and which originates from a global decrease of sulfatase function (2, C.R.B. wrote the paper. 3). The promiscuity of FGE has enabled its use in biotechnology Conflict of interest statement: C.R.B. is a cofounder and member of the Scientific Advisory and therapeutic applications, such as site-specific drug attach- Board of Redwood Bioscience (a subsidiary of Catalent, Inc.), which has exclusive rights to ment to fGly in monoclonal antibodies (4–6). the SMARTag technology based on protein modification by FGE. C.R.B. is also a cofounder Initially, FGE was thought to catalyze Cys-to-fGly conversion of Palleon Pharmaceuticals, Enable Biosciences, and InterVenn Biosciences, and a member using an unprecedented cofactorless oxidase/monooxygenase of the Board of Directors of Eli Lilly & Co. mechanism, relying on an essential redox-active cysteine pair (7). This article is a PNAS Direct Submission. J.E.P.-H. is a guest editor invited by the However, in 2015, Holder et al. (8) provided evidence for acti- Editorial Board. vation and stoichiometric Cu binding in Streptomyces coelicolor Published under the PNAS license. and human FGEs, implicating FGE as a Cu-dependent metal- Data deposition: The X-ray crystallography data have been deposited in the Protein Data loenzyme. Further investigation was carried out by Knop et al. Bank, www.wwpdb.org (PDB ID code 6MUJ). − 17 1M.J.A. and K.K.M. contributed equally to this work. (9), detailing that high-affinity (Kd ∼10 M) copper binding is dependent on the aforementioned active-site cysteine pair (10). 2Present address: Protein Chemistry, Genentech, South San Francisco, CA 94080. This was unexpected because typical mononuclear copper oxi- 3To whom correspondence may be addressed. Email: [email protected], edward. dases contain N- and O-ligands at higher coordination states [email protected], or [email protected]. (11). FGE crystal structures bound to catalytically inactive Ag(I) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and Cd(II) have provided structural evidence for a dicysteine 1073/pnas.1818274116/-/DCSupplemental. binding motif reminiscent of known Cu(I) binding proteins (12). www.pnas.org/cgi/doi/10.1073/pnas.1818274116 PNAS Latest Articles | 1of6 Downloaded by guest on September 25, 2021 HS O H treatment (SI Appendix, Fig. S4). Thus, reducing equivalents for this process ultimately originate from FGE cysteine thiols. Binding of Cu(I) to scFGE was also observed; anaerobic addition FGE, O + − 2 of one equivalent of Cu(MeCN)4 PF6 yielded stable holoenzyme N N with >0.98:1 Cu:FGE after buffer exchange. H H O O Single-Turnover Activity of Cu(I)– and Cu(II)–FGE. The catalytic Cys fGly properties of FGE in each copper oxidation state have not been addressed directly in prior studies. Although the activity of Fig. 1. The O2-dependent conversion of cysteine to Cα-formylglycine (fGly) Cu(I)-loaded FGE has been demonstrated under steady-state catalyzed by FGE. conditions (9, 10), our data prove that apo-FGE binds both Cu(I) and Cu(II). The observed rate constant for the autor- − − eductive decay of Cu(II)–scFGE (8 × 10 4 s 1) is more than two −1 direct evidence that FGE can bind both copper redox states with orders of magnitude lower than that of kcat for scFGE (0.3 s ) the same active-site ligands. By directly interrogating Cu(I)–FGE (8). Therefore, it is kinetically feasible that Cu(II)–scFGE could using X-ray absorption spectroscopy (XAS), the linear two- be catalytically competent in the presence of substrate and O2. coordinate crystallographic model was confirmed in solution. As the oxidation state of the resting enzyme places specific Moreover, these studies reveal a shift to a three-coordinate constraints on the mechanistic pathway available for substrate Tris(thiolate) Cu(I) complex upon inner-sphere substrate oxidation, explicitly testing the catalytic ability of each Cu–FGE binding, before oxygen binding and activation. Finally, in the redox state is of fundamental importance. reaction of the preformed enzyme–substrate complex with O2,a Single-turnover reactions containing 1:1 scFGE:substrate were transient intermediate is observed by stopped-flow absorbance employed to compare the activity of Cu(I)– and Cu(II)–scFGE spectroscopy. Together, these results represent the structural during the first turnover (Fig. 4). These assays used a 15-mer characterization of Cu-bound FGE in both redox states, and peptide substrate (DNP-scP15) containing a 13-residue recog- definitively identify the O2 activating site. nition motif from an S. coelicolor sulfatase and an N-terminal chromophore. To account for the inherent instability of the Results Cu(II)–scFGE complex, one equivalent of Cu(NO3)2 was added Discovery and Characterization of a Transient Cu(II)–FGE Complex. to apo-scFGE immediately before each reaction time course. Initial interrogation of the S. coelicolor FGE (scFGE) copper Within 2 min, Cu(I)–scFGE achieves nearly 70% conversion of center was pursued using UV-vis absorption and EPR (Figs.

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