Formylglycine Aldehyde Tag—Protein Engineering through a Novel Post‐translational Modification

Frese, Marc-André; Dierks, Thomas

doi:10.1002/cbic.200800801

Cited by 26 publications

(22 citation statements)

References 22 publications

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“…The main product of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 25 the reaction is N-formylglycine. There are reports on the biological activities of this species [71][72][73][74][75][76] and the transient formation of N-oxalylglycine, which is known to be an efficient enzyme inhibitor, [77][78][79][80][81][82] can also be the subject of interest. In this respect, it is important to note that Nformylglycine may accumulate in stock solutions of MCG and cause unwanted artefacts in chemical and biological studies.…”

Section: Discussionmentioning

confidence: 99%

Decomposition of N-Chloroglycine in Alkaline Aqueous Solution: Kinetics and Mechanism

Szabó

Baranyai

Somsák

et al. 2015

Chem. Res. Toxicol.

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The decomposition kinetics and mechanism of N-chloroglycine (MCG) was studied under very alkaline conditions ([OH(-)] = 0.01-0.10 M). The absorbance change is consistent with two consecutive first-order processes in the 220-350 nm wavelength range. The first reaction is linearly dependent on [OH(-)] and interpreted by the formation of a carbanion from MCG in an equilibrium step (KOH) and a subsequent loss of chloride ion from this intermediate: kobs1 = KOH k1 = (6.4 ± 0.1) × 10(-2) M(-1) s(-1), I = 1.0 M (NaClO4), and T = 25.0 °C. The second process is assigned to the first-order decomposition of N-oxalylglycine, which is also formed as an intermediate in this system: kobs2 = (1.2 ± 0.1) × 10(-3) s(-1). Systematic (1)H and (13)C NMR measurements were performed in order to identify and follow the concentration changes of the reactant, intermediate, and product. It is confirmed that the decomposition proceeds via the formation of glyoxylate ion and produces N-formylglycine as a final product. This compound is stable for an extended period of time but eventually hydrolyses into formate and glycinate ions. A detailed mechanism is postulated which resolves the controversies found in earlier literature results.

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Section: Discussionmentioning

confidence: 99%

Decomposition of N-Chloroglycine in Alkaline Aqueous Solution: Kinetics and Mechanism

Szabó

Baranyai

Somsák

et al. 2015

Chem. Res. Toxicol.

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“…81 This observation led us to speculate that the CXPXR sequence could be exploited as a means to genetically encode fGly residues in various proteins of interest. The aldehyde group, in addition to its unique catalytic nature within sulfatases, is also a reactive electrophile with orthogonal reactivity to canonical protein side chain functionalities.…”

Section: Fgly Provides a Genetically Encoded Site-specific Protein Bmentioning

confidence: 99%

Formylglycine, a Post-Translationally Generated Residue with Unique Catalytic Capabilities and Biotechnology Applications

2014

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Formylglycine (fGly) is a catalytically essential residue found almost exclusively in the active sites of type I sulfatases. Formed by post-translational oxidation of cysteine or serine side chains, this aldehyde-functionalized residue participates in a unique and highly efficient catalytic mechanism for sulfate ester hydrolysis. The enzymes that produce fGly, formylglycine-generating enzyme (FGE) and anaerobic sulfatase-maturating enzyme (anSME), are as unique and specialized as fGly itself. FGE especially is structurally and mechanistically distinct, and serves the sole function of activating type I sulfatase targets. This review summarizes the current state of knowledge regarding the mechanism by which fGly contributes to sulfate ester hydrolysis, the molecular details of fGly biogenesis by FGE and anSME, and finally, recent biotechnology applications of fGly beyond its natural catalytic function.

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“…This short amino acid sequence (defined by the broader consensus sequence CxPxR) is recognized by the FGE, which catalyzes the co-translational oxidation of the cysteine residue to an aldehyde-bearing Cα-formylglycine (FGly) residue 11–13 , a modification that is required for sulfatase activity. Type 1 sulfatases are found in most organisms, and likewise the amino acid modification and FGEs that generate it occur in all domains of life 14 .…”

Section: Introductionmentioning

confidence: 99%

Site-specific chemical protein conjugation using genetically encoded aldehyde tags

et al. 2012

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We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with α-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.

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Formylglycine Aldehyde Tag—Protein Engineering through a Novel Post‐translational Modification

Cited by 26 publications

References 22 publications

Decomposition of N-Chloroglycine in Alkaline Aqueous Solution: Kinetics and Mechanism

Decomposition of N-Chloroglycine in Alkaline Aqueous Solution: Kinetics and Mechanism

Formylglycine, a Post-Translationally Generated Residue with Unique Catalytic Capabilities and Biotechnology Applications

Site-specific chemical protein conjugation using genetically encoded aldehyde tags

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