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广东快乐十分任2计划:Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation
广东快乐十分投注下载 www.hmclip.net 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)
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Many enzymes harness the energy of molecular oxygen to catalyze diverse transformations. However, reaction with oxygen is kinetically challenging and must be precisely controlled to prevent cellular damage. The formylglycine-generating enzyme activates its targets by utilizing a mononuclear copper center to facilitate oxidation of a substrate cysteine. We reveal the structure of its atypical copper active site and discover that substrate binding directly to copper precedes and initiates reactivity toward oxygen. We furthermore identify a transient intermediate that may provide evidence for the involvement of an elusive catalytic species. This work uncovers a distinct strategy among copper enzymes that exploits a dynamic binding site to tightly couple the activation of oxygen with selective substrate oxidation.
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.
?1M.J.A. and K.K.M. contributed equally to this work.
?2Present address: Protein Chemistry, Genentech, South San Francisco, CA 94080.
- ?3To whom correspondence may be addressed. Email: , , or .
Author contributions: M.J.A., K.K.M., J.L.-V., J.A.T., E.I.S., and C.R.B. designed research; 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 C.R.B. wrote the paper.
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.
This article is a PNAS Direct Submission. J.E.P.-H. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1818274116/-/DCSupplemental.
Published under the PNAS license.