Characterization of Dye-Decolorizing Peroxidases from Rhodococcus jostii RHA1
Characterization of Dye-Decolorizing Peroxidases from Rhodococcus jostii RHA1
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DOI：
10.1021/bi200427h
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发表时间：
2011-06-14
期刊：
影响因子：
2.9
通讯作者：
Eltis, Lindsay D.
中科院分区：
文献类型：
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作者：
Roberts, Joseph N.;Singh, Rahul;Eltis, Lindsay D.
土壤细菌Jostii RHA1含有两个染料脱色过氧化物酶(DYPs)，根据它们所代表的亚家族命名：DypA，预测为周质；DypB，与木质素降解有关。对这些酶的稳态动力学研究表明，它们的过氧化物酶活性比C型和D型DYPs低得多。然而，DypA对活性艳蓝4(k(CAT)/K-m=12800+/-600M-1 S(-1))的明显特异性是ABTS或邻苯三酚的6倍，这与以前的DYP特征一致。相反，DypB对ABTS(k(CAT)/K-m=2000+/-100M-1 S(-1))和氧化型Mn-II(k(CAT)/K-m=25.1+/-0.1M-1 S(-1))显示出最明显的特异性。电子顺磁共振(EPR)谱检测到进一步的差异：当两个DYP在静止状态下都含有高自旋(S=S/2)Fe-III时，DypA有一个菱形的高自旋信号(g(Y)=6.32，g(X)=5.45，g(Z)=1.97)，而DypB有一个主要的轴向信号(g(Y)=6.09，g(X)=5.45，g(Z)=1.99)。DypA与H_2O_2反应生成具有化合物II(Fe-IV=O)特征的中间体。而DypB与H_2O_2的二级反应速率常数为(1.79+/-0.06)×10~(5)M~(-1)S(~(-1))，生成相对稳定的绿色中间体(t(1/2)类似于9min)。虽然该中间体的电子吸收光谱与植物型过氧化物酶的化合物I相似，但其EPR谱与弱偶联的蛋白质自由基的EPR谱比与[Fe-IV=O Por(中心点)](+)物种的EPR谱更一致。DypB的X-射线晶体结构测定到1.4埃分辨率，显示出与组氨酸形成六配位的血红素铁和占据轴向位置的溶剂物种。一个溶剂通道潜在地为过氧化氢提供了通往血红素远端表面的通道。一个浅口袋将丙酸亚铁血红素暴露在溶剂中，并含有一簇可能结合锰-II的酸性残基。深入了解DypB的结构和功能有助于其工程改进木质纤维素的降解。
The soil bacterium Rhodococcus jostii RHA1 contains two dye-decolorizing peroxidases (DyPs) named according to the subfamily they represent: DypA, predicted to be periplasmic, and DypB, implicated in lignin degradation. Steady-state kinetic studies of these enzymes revealed that they have much lower peroxidase activities than C- and D-type DyPs. Nevertheless, DypA showed 6-fold greater apparent specificity for the anthraquinone dye Reactive Blue 4 (k(cat)/K-m = 12800 +/- 600 M-1 s(-1) than either ABTS or pyrogallol, consistent with previously characterized DyPs. By contrast, DypB showed the greatest apparent specificity for ABTS (k(cat)/K-m = 2000 +/- 100 M-1 s(-1)) and also oxidized Mn-II (k(cat)/K-m = 25.1 +/- 0.1 M-1 s(-1)). Further differences were detected using electron paramagnetic resonance (EPR) spectroscopy: while both DyPs contained high-spin (S = s/2) Fe-III in the resting state, DypA had a rhombic high-spin signal (g(y) = 6.32, g(x) = 5.45, and g(z) = 1.97) while DypB had a predominantly axial signal (g(y) = 6.09, g(x) = 5.45, and g(z) = 1.99). Moreover, DypA reacted with H2O2 to generate an intermediate with features of compound II (Fe-IV=O). By contrast, DypB reacted with H2O2 with a second-order rate constant of (1.79 +/- 0.06) x 10(5) M-1 s(-1) to generate a relatively stable green-colored intermediate (t(1/2) similar to 9 min). While the electron absorption spectrum of this intermediate was similar to that of compound I of plant-type peroxidases, its EPR spectrum was more consistent with a poorly coupled protein-based radical than with an [Fe-IV=O Por(center dot)](+) species. The X-ray crystal structure of DypB, determined to 1.4 angstrom resolution, revealed a hexacoordinated heme iron with histidine and a solvent species occupying axial positions. A solvent channel potentially provides access to the distal face of the heme for H2O2. A shallow pocket exposes heme propionates to the solvent and contains a cluster of acidic residues that potentially bind Mn-II. Insight into the structure and function of DypB facilitates its engineering for the improved degradation of lignocellulose.