Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

Holder, Patrick G.; Jones, Lesley C.; Drake, Penelope M.; Barfield, Robyn M.; Bañas, Stefanie; Hart, Gregory W. de; Baker, J. L.; Rabuka, David

doi:10.1074/jbc.m115.652669

Cited by 59 publications

(60 citation statements)

References 52 publications

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“…While this manuscript was under review,a ni ndependent study,d escribing as imilar activating effect of copper on FGE from humansa nd from Streptomyces coelicolor,w as published in the Journalo fB iological Chemistry.T he two studies demonstrate the generality of copper-dependent activation of in vitro FGE activity. [16]…”

Section: Af Inal Notementioning

confidence: 99%

In Vitro Reconstitution of Formylglycine‐Generating Enzymes Requires Copper(I)

et al. 2015

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Formylglycine-generating enzymes (FGEs) catalyze O2 -dependent conversion of specific cysteine residues of arylsulfatases and alkaline phosphatases into formylglycine. The ability also to introduce unique aldehyde functions into recombinant proteins makes FGEs a powerful tool for protein engineering. One limitation of this technology is poor in vitro activity of reconstituted FGEs. Although FGEs have been characterized as cofactor-free enzymes we report that the addition of one equivalent of Cu(I) increases catalytic efficiency more than 20-fold and enables the identification of stereoselective C-H bond cleavage at the substrate as the rate-limiting step. These findings remove previous limitations of FGE-based protein engineering and also pose new questions about the catalytic mechanism of this O2 -utilizing enzyme.

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Section: Af Inal Notementioning

confidence: 99%

In Vitro Reconstitution of Formylglycine‐Generating Enzymes Requires Copper(I)

et al. 2015

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“…[17] This recognition sequence can be introduced into recombinant proteins and converted by FGE in vivo or in vitro. [19][20][21][22] In addition, the iron-sulfur protein AtsB of the radical S-adenosyl methionine (radical-SAM) protein superfamily is another FGly-generating system that is exclusively found in prokaryotes. [19][20][21][22] In addition, the iron-sulfur protein AtsB of the radical S-adenosyl methionine (radical-SAM) protein superfamily is another FGly-generating system that is exclusively found in prokaryotes.…”

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confidence: 99%

Two‐fold Bioorthogonal Derivatization by Different Formylglycine‐Generating Enzymes

et al. 2018

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Formylglycine-generating enzymes are of increasing interest in the field of bioconjugation chemistry. They catalyze the site-specific oxidation of a cysteine residue to the aldehyde-containing amino acid C -formylglycine (FGly). This non-canonical residue can be generated within any desired target protein and can subsequently be used for bioorthogonal conjugation reactions. The prototypic formylglycine-generating enzyme (FGE) and the iron-sulfur protein AtsB display slight variations in their recognition sequences. We designed specific tags in peptides and proteins that were selectively converted by the different enzymes. Combination of the different tag motifs within a single peptide or recombinant protein enabled the independent and consecutive introduction of two formylglycine residues and the generation of heterobifunctionalized protein conjugates.

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“…The catalytic mechanism of FGE is not yet fully understood. Recent work, however, showed that FGE is a copper‐dependent oxidase that might take an approach to O 2 activation and C−H cleavage similar to that of the copper‐dependent lytic polysaccharide monooxygenases (LPMOs), the peptidylglycine α‐hydroxylating monooxygenase, or dopamine/tyramine‐β‐monooxygenases . In the course of isolating the catalytic principle of FGE from T. curvata we constructed a variant in which all cysteine residues outside the active site were mutated .…”

Section: Resultsmentioning

confidence: 99%

“…Because the phenol function of Tyr273 does not directly contribute to catalysis we considered alternative roles for this residue. Tyr273 could be a copper ligand and might contribute to the high Cu I affinity . Indeed, tyrosine residues do function as copper ligands in other mononuclear copper enzymes such as galactose oxidase, copper amine oxidases, and type‐AA9 LPMOs .…”

Section: Resultsmentioning

confidence: 99%

Mutation of Conserved Residues Increases in Vitro Activity of the Formylglycine‐Generating Enzyme

2017

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The formylglycine-generating enzyme (FGE) recognizes proteins with a specific cysteine-containing six-amino-acid motif and converts this cysteine residue into formylglycine. The resulting aldehyde function provides a unique handle for selective protein labeling. We have identified two mutations in FGE from Thermomonospora curvata that increase this catalytic efficiency more than 40-fold. The resulting activity and stability, as well as its ease of recombinant production, make this FGE variant a practical reagent for in vitro protein engineering.

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Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

Cited by 59 publications

References 52 publications

In Vitro Reconstitution of Formylglycine‐Generating Enzymes Requires Copper(I)

In Vitro Reconstitution of Formylglycine‐Generating Enzymes Requires Copper(I)

Two‐fold Bioorthogonal Derivatization by Different Formylglycine‐Generating Enzymes

Mutation of Conserved Residues Increases in Vitro Activity of the Formylglycine‐Generating Enzyme

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