Rahmanpour Rahman, Rea Dean, Jamshidi Shirin, Fülöp Vilmos, Bugg Timothy D H
Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK.
School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK.
Arch Biochem Biophys. 2016 Mar 15;594:54-60. doi: 10.1016/j.abb.2016.02.019. Epub 2016 Feb 18.
A Dyp-type peroxidase enzyme from thermophilic cellulose degrader Thermobifida fusca (TfuDyP) was investigated for catalytic ability towards lignin oxidation. TfuDyP was characterised kinetically against a range of phenolic substrates, and a compound I reaction intermediate was observed via pre-steady state kinetic analysis at λmax 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a β-aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8 Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, positioned close to active site residues Asp-203 and Arg-315. The structure contains two channels providing access to the heme cofactor for organic substrates and hydrogen peroxide. Site-directed mutant D203A showed no activity towards phenolic substrates, but reduced activity towards ABTS, while mutant R315Q showed no activity towards phenolic substrates, nor ABTS.
对嗜热纤维素降解菌栖热放线菌（Thermobifida fusca）中的一种DyP型过氧化物酶（TfuDyP）进行了研究，以考察其对木质素氧化的催化能力。通过对一系列酚类底物进行动力学表征，利用预稳态动力学分析在404 nm最大吸收波长处观察到了化合物I反应中间体。TfuDyP对牛皮纸木质素具有反应活性，并且发现它能氧化一种β-芳基醚木质素模型化合物，形成一种氧化二聚体。测定了TfuDyP的晶体结构，分辨率为1.8 Å，发现其血红素中心结合有一个双原子氧配体，该配体靠近活性位点残基天冬氨酸-203（Asp-203）和精氨酸-315（Arg-315）。该结构包含两个通道，为有机底物和过氧化氢提供通向血红素辅因子的途径。定点突变体D203A对酚类底物无活性，但对ABTS的活性降低，而突变体R315Q对酚类底物和ABTS均无活性。
