An artificial di-iron oxo-protein with phenol oxidase activity

Faiella, Marina; Andreozzi, Concetta; Rosales, Rafael T. M. de; Pavone, Vincenzo; Maglio, Ornella; Nastri, Flavia; DeGrado, William F.; Lombardi, Angela

doi:10.1038/nchembio.257

Cited by 175 publications

(179 citation statements)

References 25 publications

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“…In the pH range 6-7.4, only two singlets (∼7.0 and 7.7 ppm) are observed in D 2 O for apo-(TRIL23H) 3 , corresponding to the three equivalent His H δ and H e protons, respectively. The addition of 1 eq of [Cu(I)(CH 3 CN) 4 ] BF 4 to apo-(TRIL23H) 3 at pH 6 or pH 7.4 leads to changes in the NMR spectra with the appearance of multiple peaks in the range 6.6-8.7 ppm (Fig. S1 A-D).…”

Section: Resultsmentioning

confidence: 99%

“…For these two samples, the amount of trapped NO is 71% and 48% after 1 h, respectively, of that from a control sample containing [Cu(I)(CH 3 CN) 4 ] + .…”

Section: +mentioning

confidence: 99%

“…Three important examples of functional, de novo designed enzymes are the Dueferri, MID1-zinc, and zinc(II) TRI systems. The first uses Fe bound to a four-helix bundle in a redox role to oxidize phenols (3,4), whereas the other two contain zinc(II) in a (His) 3 environment and are models for carbonic anhydrase (5,6). The zinc(II) TRI system, in particular, exploits Zn(II) to mimic very well the active site structure and CO 2 -hydrolytic chemistry of the native enzyme, using three-stranded coiled coils (6).…”

Section: +mentioning

confidence: 99%

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Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

Tegoni

Yu²,

Bersellini

et al. 2012

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

101

188

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One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H) 3 +/2+ , where Cu(I/II) is embeded in α-helical coiled coils, as a model for the Cu T2 center of copper nitrite reductase. In Cu(I/II)(TRIL23H) 3 +/2+ , Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His) 3 active site is characterized in both oxidation states, revealing similarities to the Cu T2 site in the natural enzyme. The species Cu(II)(TRIL23H) 3 2+ in aqueous solution can be reduced to Cu(I)(TRIL23H) 3 + using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H) 3 2+ acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed.

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Section: Resultsmentioning

confidence: 99%

“…For these two samples, the amount of trapped NO is 71% and 48% after 1 h, respectively, of that from a control sample containing [Cu(I)(CH 3 CN) 4 ] + .…”

Section: +mentioning

confidence: 99%

Section: +mentioning

confidence: 99%

See 1 more Smart Citation

Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

Tegoni

Yu²,

Bersellini

et al. 2012

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

101

188

View full text Add to dashboard Cite

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“…[21][22][23][24] The function of a family of de novo proteins, di-iron oxo-proteins or ''Due Ferri'' proteins, 25 was altered through modifications of a loop far from the active site, mutations chosen to redirect the enzyme's intended function. 26 The addition of an extra metal-binding residue and adjusting the substrate cavity switch a due ferri protein originally intended to oxidize hydroquinones to catalyze the selective N-hydroxylation of arylamines. 27 While design efforts focused on metal-free enzymes have yielded many useful in silico tools, these tools are insufficient for metal-containing enzymes.…”

Section: Introductionmentioning

confidence: 99%

Predictive methods for computational metalloenzyme redesign – a test case with carboxypeptidase A

Valdez

Morgenstern

Eberhart

et al. 2016

Phys. Chem. Chem. Phys.

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“…156,157 Control over helix orientation is generally provided by covalent linkage of helix termini through disulfide bonds 154,158 or short polypeptide linkers. [159][160][161] Functionality is incorporated by including residues within the core of the coiled coil that bind redox-active metal ions or cofactors. 154,158,[160][161][162][163] Membrane association and integration may also be designed into these maquettes.…”

Section: 150mentioning

confidence: 99%

Bioproduction and self-assembly of designer peptides

Fletcher¹

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Self-assembly is a powerful method for producing controlled morphologies at the nanometre scale. Peptides are biomolecules capable of self-assembly and have the potential for use in a variety of applications such as emulsion and foam stabilisation, wound healing and drug delivery. The investigation of peptide sequence-structure relationships is a rapidly advancing field of study, which in many cases now allows the targeted design of self-assembling peptides.While self-assembling peptides show potential in several fields, their large-scale adoption is limited by the high production expenses associated with conventional solid-phase synthesis. A potentially cheaper and more renewable approach to peptide production is bacterial expression. However, the bioproduction of peptides is a non-trivial process, and generally involves the expression of peptides as part of a fusion protein in which the target peptide is only a small portion of the expressed product. An alternate approach involves peptide concatemers, in which the target peptide makes up the majority of the expressed construct.The work reported in this thesis focused on the design, characterisation and bioproduction of self-assembling α-helical peptides. It was particularly focussed on the development of amphiphillic α-helical peptides with applications as surfactants and hydrogelators. The aim of this work was to further the field of de novo peptide design, while also investigating an approach for peptide bioproduction. This work aimed to combine the requirements for peptide bioproduction with end-use functionality.Chapter 2 details successful bioproduction of an anionic helical surfactant peptide EDP-11, as part of a charge-paired heteroconcatemer with the cationic expression partner RDP-4. The method utilised designed assembly of the constituent α-helical peptides to generate a stably folded coiled-coil concatemer. The polypeptide sequence was further optimized for molecular charge, hydropathy and predicted protease resistance. This process allowed expression of a soluble concatemer that accumulated to high levels (22% of total protein) in E. coli. The expressed concatemer possessed extreme stability to heat and proteases, allowing isolation by simple heat and pH precipitation, yielding concatemer at 130 mg per gram of dry cell weight and >99% purity. Key parameters used in designing the heteroconcatemer were then compared to those of all open reading frames of several reference ii proteomes in an attempt to gain insight into the mechanistic basis for the high stability of the designed miniprotein.Following bioproduction using this concatemer approach, further processing was required to produce monomeric peptides for use in surfactant applications. The design of acid-cleavable aspartate-proline sites within the concatemer sequence allowed for simple heat-and acid-mediated cleavage to give constituent peptides.Chapter 3 details characterization of cleavage of the expressed concatemer by coupled liquid chromatography-mass spectrometry and includes mod...

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An artificial di-iron oxo-protein with phenol oxidase activity

Cited by 175 publications

References 25 publications

Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

Predictive methods for computational metalloenzyme redesign – a test case with carboxypeptidase A

Bioproduction and self-assembly of designer peptides

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