4W7M

CRYSTAL STRUCTURE OF A DECOLORIZING PEROXIDASE (DYP) FROM AURICULARIA AURICULA-JUDAE. W377S MUTANT

4W7M の概要

• エントリーDOI: 10.2210/pdb4w7m/pdb
• 分子名称: Dye-decolorizing peroxidase, PROTOPORPHYRIN IX CONTAINING FE (3 entities in total)
• 機能のキーワード: oxidoreductase, heme, glycoprotein
• 由来する生物種: Auricularia auricula-judae (ear fungus)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 95249.32

構造登録者

Medrano, F.J.,Romero, A. (登録日: 2014-08-22, 公開日: 2015-03-11, 最終更新日: 2024-01-10)

主引用文献

Linde, D.,Pogni, R.,Canellas, M.,Lucas, F.,Guallar, V.,Baratto, M.C.,Sinicropi, A.,Saez-Jimenez, V.,Coscolin, C.,Romero, A.,Medrano, F.J.,Ruiz-Duenas, F.J.,Martinez, A.T.
Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study.
Biochem.J., 466:253-262, 2015

PubMed Abstract: Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H₂O₂-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (k(cat) > 200 s⁻¹) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 k(cat) ~20 s⁻¹) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant.

PubMed: 25495127
DOI: 10.1042/BJ20141211

実験手法

X-RAY DIFFRACTION (1.15 Å)