Structural Basis of Substrate Conversion in a New Aromatic Peroxygenase

Piontek, Klaus; Strittmatter, Eric F.; Ullrich, René; Gröbe, Glenn; Pecyna, Marek J.; Kluge, Martin; Scheibner, Katrin; Hofrichter, Martin; Plattner, Dietmar A.

doi:10.1074/jbc.m113.514521

Cited by 128 publications

(143 citation statements)

References 54 publications

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“…S. cerevisiae tends to hyperglycosylate foreign proteins up to levels of ϳ50%, conferring on them increased stability and protection against proteolytic degradation. The glycosylation of wt UPO1 observed was exclusively dependent on 6 predicted N-glycosylation sites (Oglycosylation sites are not described for the enzyme), associated with up to 8 moieties of the high-mannose type (30,35). None of the amino acid substitutions in PaDa-I introduced new glycosylation motifs, and thus, the higher sugar content in the mutant may be due to an increased Golgi residence time that leads to the addition of more mannose moieties, as described in other directedevolution studies in yeast (14,17).…”

Section: Resultsmentioning

confidence: 98%

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Molina‐Espeja

García-Ruiz

González-Pérez

et al. 2014

Appl Environ Microbiol

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c Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the k cat /K m value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

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Section: Resultsmentioning

confidence: 98%

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Molina‐Espeja

García-Ruiz

González-Pérez

et al. 2014

Appl Environ Microbiol

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200

281

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“…[26] Thus, we examined the recent AaeUPO1 crystal structure to find a means to suppress alternative peroxidative pathways. [27] QM/MM calculations identified Trp24 as the highest oxidizable surface residue (see computational analysis below); hence, we constructed site-directed variants using the PaDa-I and JaWa variants as templates. The corresponding PaDa-I-W24F and JaWa-W24F mutants were compared: Irrespective of the variant and the reducing substrate tested, the W24F mutation decreased the peroxidative activity by ~60% ( Figure 3B).…”

Section: Biochemical Characterizationmentioning

confidence: 99%

“…The structure of wild-type UPO1 (purified from A. aegerita culture) at a resolution of 2.1 Å (Protein Data Bank Europe [PDB] accession number 2YOR) was used as departure point for PaDa-I and JaWa variants modeling. [27] The wild-type UPO1 crystal was used. Five different mutations (V57A-L67F-V75I-I248V-F311L) were introduced to model PaDa-I variant, with two additional mutations (G241D-R257K) for JaWa.…”

Section: Computational Analysismentioning

confidence: 99%

Synthesis of 1‐Naphthol by a Natural Peroxygenase Engineered by Directed Evolution

et al. 2016

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Abstract:There is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular fungal aromatic peroxygenase with mono(per)oxygenase activity was engineered to selectively convert naphthalene into 1-naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene while quenching the undesired peroxidative activity on 1-naphthol (one-electron oxidation). The resulting double mutant (G241D-R257K) of this process was characterized biochemically and computationally. The conformational changes produced by directed evolution improved the substrate´s catalytic position. Powered exclusively by catalytic concentrations of H2O2, this soluble and stable biocatalyst has total turnover numbers of 50,000, with high regioselectivity (97%) and reduced peroxidative activity.

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“…Accordingly, we anticipated that these water-soluble nitroxyl radicals might be able to reduce APO-I readily but would not reduce APO-II efficiently. Furthermore, since cyclohexane carboxylic acid was found to be an excellent substrate for APO (29), the similar molecular topographies of 4-carboxy-TEMPO and 3-carboxy-PROXYL suggested that they would fit into the relatively small, conical active site of APO (25). We tested the reaction of 3-carboxy-PROXYL with the well-studied enzyme CPO in a single-turnover experiment.…”

Section: Significancementioning

confidence: 99%

“…These proteins are unrelated to CYP enzymes, according to their amino acid sequences, and are only distantly related to CPO, with about 30% sequence similarity and a similar tertiary structure (25). APO proteins have shown high activity for the oxygenation of aliphatic and aromatic hydrocarbons and a variety of other organic substrates, in sharp contrast to CPO (26).…”

mentioning

confidence: 99%

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

Wang

Ullrich

Hofrichter

et al. 2015

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

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A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapidmixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pHjump spectrophotometry revealed a basic pK a of 10.0 for the Fe IV −O−H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C−H and phenolic substrates, with second-order rate constants for C−H and O−H bond scission in the range of 10-10 7 M −1 ·s −1 . Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C−H and O−H bond scission.

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Structural Basis of Substrate Conversion in a New Aromatic Peroxygenase

Cited by 128 publications

References 54 publications

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Synthesis of 1‐Naphthol by a Natural Peroxygenase Engineered by Directed Evolution

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

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