Crystal structure of the precursor of galactose oxidase: An unusual self-processing enzyme

Firbank, S.J.; Rogers, Melanie S.; Wilmot, Carrie M.; Dooley, David M.; Halcrow, Malcolm A.; Knowles, Peter F.; McPherson, Michael J.; Phillips, Simon E. V.

doi:10.1073/pnas.231463798

Cited by 112 publications

(82 citation statements)

References 44 publications

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“…Delocalization of the tyrosyl radical onto the thioether bridge, which is predicted by density functional theory (DFT) computations (7,8) and EPR studies of copper-free GO (9), is postulated to contribute to the energetic stabilization of GO oxy . This postulate is consistent with the in-plane conformation of the cysteine-tyrosine cross-link observed in all known crystal structures of GO (3)(4)(5), yet the extent of hole delocalization on sulfur and consequent energetic influence in copper-bonded phenoxyls including GO oxy remain unknown (9,10).…”

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confidence: 83%

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Sulfanyl stabilization of copper-bonded phenoxyls in model complexes and galactose oxidase

Verma

Pratt

Storr

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Integrating sulfanyl substituents into copper-bonded phenoxyls significantly alters their optical and redox properties and provides insight into the influence of cysteine modification of the tyrosine cofactor in the enzyme galactose oxidase. The model complexes ½1 SR2 þ are class II mixed-valent Cu II -phenoxyl-phenolate species that exhibit intervalence charge transfer bands and intense visible sulfur-aryl π → π Ã transitions in the energy range, which provides a greater spectroscopic fidelity to oxidized galactose oxidase than non-sulfur-bearing analogs. The potentials for phenolate-based oxidations of the sulfanyl-substituted 1 SR2 are lower than the alkylsubstituted analogs by up to ca. 150 mV and decrease following the steric trend: −S t Bu > −S i Pr > −SMe. Density functional theory calculations suggest that reducing the steric demands of the sulfanyl substituent accommodates an in-plane conformation of the alkylsulfanyl group with the aromatic ring, which stabilizes the phenoxyl hole by ca. 8 kcal mol −1 (1 kcal ¼ 4.18 kJ; 350 mV) through delocalization onto the sulfur atom. Sulfur K-edge X-ray absorption spectroscopy clearly indicates a contribution of ca. 8-13% to the hole from the sulfur atoms in ½1 SR2 þ . The electrochemical results for the model complexes corroborate the ca. 350 mV (density functional theory) contribution of hole delocalization on to the cysteine-tyrosine cross-link to the stability of the phenoxyl radical in the enzyme, while highlighting the importance of the in-plane conformation observed in all crystal structures of the enzyme.Marcus-Hush analysis | density functional theory reduction potentials | noninnocent ligands | magnetic coupling G alactose oxidase (GO) is an enzyme that selectively oxidizes primary alcohols to aldehydes via a unique Cu II -tyrosyl radical with concomitant reduction of dioxygen to hydrogen peroxide (1, 2). The active site of GO contains a Cu center ligated by two histidines, one unmodified tyrosine (axial) and one tyrosine residue (equatorial) that is covalently cross-linked to a cysteine residue in a posttranslational oxidative modification step (3-5) (Fig. 1). The consensus mechanism involves two catalytically relevant forms: the reduced (GO red ), which contains a Cu I -tyrosine unit, and the oxidized (GO oxy ), which contains a Cu II center and a cysteine-modified tyrosyl radical. One-electron reduction of GO oxy generates the inactive semireduced (GO semi ) form, which contains a Cu II -tyrosinate unit. Cysteine-modification of the redox-active tyrosine significantly influences the redox properties of the active oxidant: The potential of the Tyr-Cys/Tyr•-Cys couple estimated from the GO semi ∕ GO oxy interconversion is ca. 400 mV [vs. normal hydrogen electrode (NHE), pH 7.5], which is significantly lower than free tyrosine in solution (ca. 900 mV) and unmodified redox-active tyrosines in other enzymes (ca. 660-1,000 mV) (1, 6). Delocalization of the tyrosyl radical onto the thioether bridge, which is predicted by density functional theory (DFT) compu...

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confidence: 83%

“…The active site of GO contains a Cu center ligated by two histidines, one unmodified tyrosine (axial) and one tyrosine residue (equatorial) that is covalently cross-linked to a cysteine residue in a posttranslational oxidative modification step (3)(4)(5) (Fig. 1).…”

mentioning

confidence: 99%

Sulfanyl stabilization of copper-bonded phenoxyls in model complexes and galactose oxidase

Verma

Pratt

Storr

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…There is precedent for the participation of cysteine residues in the formation of amino acid cross-links that are stable under reducing conditions. For example, a cysteine residue located in the active site of galactose oxidase has been demonstrated to participate in a thioether linkage with a nearby tyrosine, thus forming an intramolecular amino acid cross-link (10). Given the marked decrease in the electrophoretic mobility of the modified form of Keap1, this modified form of Keap1 may result from an intermolecular protein (15,19,25,29,37) have illuminated one important mechanism for Keap1-mediated repression and the ability of chemical inducers to enable Nrf2 to escape Keap1-mediated repression.…”

Section: Discussionmentioning

confidence: 99%

Distinct Cysteine Residues in Keap1 Are Required for Keap1-Dependent Ubiquitination of Nrf2 and for Stabilization of Nrf2 by Chemopreventive Agents and Oxidative Stress

Zhang

Hannink

2003

Molecular and Cellular Biology

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1,277

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A common feature of diverse chemopreventive agents is the ability to activate expression of a genetic program that protects cells from reactive chemical species that, if left unchecked, would cause mutagenic DNA damage. The bZIP transcription factor Nrf2 has emerged as a key regulator of this cancer-preventive genetic program. Nrf2 is normally sequestered in the cytoplasm by a protein known as Keap1. Chemopreventive agents allow Nrf2 to escape from Keap1-mediated repression, although the molecular mechanism(s) responsible for activation of Nrf2 is not understood. In this report, we demonstrate that Keap1 does not passively sequester Nrf2 in the cytoplasm but actively targets Nrf2 for ubiquitination and degradation by the proteosome under basal culture conditions. We have identified two critical cysteine residues in Keap1, C273 and C288, that are required for Keap1-dependent ubiquitination of Nrf2. Both sulforaphane, a chemopreventive isothiocyanate, and oxidative stress enable Nrf2 to escape Keap1-dependent degradation, leading to stabilization of Nrf2, increased nuclear localization of Nrf2, and activation of Nrf2-dependent cancer-protective genes. We have identified a third cysteine residue in Keap1, C151, that is uniquely required for inhibition of Keap1-dependent degradation of Nrf2 by sulforaphane and oxidative stress. This cysteine residue is also required for a novel posttranslational modification to Keap1 that is induced by oxidative stress. We propose that Keap1 is a component of a novel E3 ubiquitin ligase complex that is specifically targeted for inhibition by both chemopreventive agents and oxidative stress.

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“…Nevertheless, some surprising examples of unexpected crosslinks have been brought to light by protein structure analysis or by the observation of unusual spectroscopic or biophysical properties. Examples include the Cys-Tyr bond in galactose oxidase (1), which provides a radical center; similar bonds in some catalases (2); the His-Tyr bond in cytochrome C oxidase (3); and the remarkable chromophore of GFP (4). These, and other examples, arise through intramolecular reactions facilitated by particular local environments.…”

mentioning

confidence: 99%

Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin

Kwon

Squire

Young

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Gram-positive bacteria are decorated by a variety of proteins that are anchored to the cell wall and project from it to mediate colonization, attachment to host cells, and pathogenesis. These proteins, and protein assemblies, such as pili, are typically long and thin yet must withstand high levels of mechanical stress and proteolytic attack. The recent discovery of intramolecular isopeptide bond cross-links, formed autocatalytically, in the pili from Streptococcus pyogenes has highlighted the role that such crosslinks can play in stabilizing such structures. We have investigated a putative cell-surface adhesin from Clostridium perfringens comprising an N-terminal adhesin domain followed by 11 repeat domains. The crystal structure of a two-domain fragment shows that each domain has an IgG-like fold and contains an unprecedented ester bond joining Thr and Gln side chains. MS confirms the presence of these bonds. We show that the bonds form through an autocatalytic intramolecular reaction catalyzed by an adjacent His residue in a serine protease-like mechanism. Two buried acidic residues assist in the reaction. By mutagenesis, we show that loss of the ester bond reduces the thermal stability drastically and increases susceptibility to proteolysis. As in pilin domains, the bonds are placed at a strategic position joining the first and last strands, even though the Ig fold type differs. Bioinformatic analysis suggests that similar domains and ester bond cross-links are widespread in Gram-positive bacterial adhesins. intramolecular ester bond | protein stability A striking feature of globular proteins is that despite the chemical diversity inherent in the side chains of their constituent amino acids, chemical reactions between these side chains are very rare. This may be explained by evolutionary selection, which minimizes reactions that could prejudice proper protein folding. Thus, the only common example of a covalent cross-link between protein side chains is the disulfide bond, which forms only in an appropriate redox environment when two Cys residues are brought together by protein folding. Nevertheless, some surprising examples of unexpected crosslinks have been brought to light by protein structure analysis or by the observation of unusual spectroscopic or biophysical properties. Examples include the Cys-Tyr bond in galactose oxidase (1), which provides a radical center; similar bonds in some catalases (2); the His-Tyr bond in cytochrome C oxidase (3); and the remarkable chromophore of GFP (4). These, and other examples, arise through intramolecular reactions facilitated by particular local environments.The recent discovery of isopeptide bonds joining Lys and Asn side chains in the proteins that make up pili on the Gram-positive bacterium Streptococcus pyogenes (5), as well as on other Gram-positive pathogens (6), has highlighted a class of proteins in which intramolecular cross-links seem to be remarkably prevalent. It includes not only Gram-positive pili but a number of other cell surface adhesins, known as mi...

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Crystal structure of the precursor of galactose oxidase: An unusual self-processing enzyme

Cited by 112 publications

References 44 publications

Sulfanyl stabilization of copper-bonded phenoxyls in model complexes and galactose oxidase

Sulfanyl stabilization of copper-bonded phenoxyls in model complexes and galactose oxidase

Distinct Cysteine Residues in Keap1 Are Required for Keap1-Dependent Ubiquitination of Nrf2 and for Stabilization of Nrf2 by Chemopreventive Agents and Oxidative Stress

Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin

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