Department of Chemistry, University of Warwick, Coventry, UK.
School of Life Sciences, University of Warwick, Coventry, UK.
FEBS J. 2018 May;285(9):1684-1700. doi: 10.1111/febs.14437. Epub 2018 Mar 30.
The identification of enzymes responsible for oxidation of lignin in lignin-degrading bacteria is of interest for biotechnological valorization of lignin to renewable chemical products. The genome sequences of two lignin-degrading bacteria, Ochrobactrum sp., and Paenibacillus sp., contain no B-type DyP peroxidases implicated in lignin degradation in other bacteria, but contain putative multicopper oxidase genes. Multi-copper oxidase CueO from Ochrobactrum sp. was expressed and reconstituted as a recombinant laccase-like enzyme, and kinetically characterized. Ochrobactrum CueO shows activity for oxidation of β-aryl ether and biphenyl lignin dimer model compounds, generating oxidized dimeric products, and shows activity for oxidation of Ca-lignosulfonate, generating vanillic acid as a low molecular weight product. The crystal structure of Ochrobactrum CueO (OcCueO) has been determined at 1.1 Å resolution (PDB: 6EVG), showing a four-coordinate mononuclear type I copper center with ligands His495, His434 and Cys490 with Met500 as an axial ligand, similar to that of Escherichia coli CueO and bacterial azurin proteins, whereas fungal laccase enzymes contain a three-coordinate type I copper metal center. A trinuclear type 2/3 copper cluster was modeled into the active site, showing similar structure to E. coli CueO and fungal laccases, and three solvent channels leading to the active site. Site-directed mutagenesis was carried out on amino acid residues found in the solvent channels, indicating the importance for residues Asp102, Gly103, Arg221, Arg223, and Asp462 for catalytic activity. The work identifies a new bacterial multicopper enzyme with activity for lignin oxidation, and implicates a role for bacterial laccase-like multicopper oxidases in some lignin-degrading bacteria.
Structural data are available in the PDB under the accession number 6EVG.
鉴定负责木质素降解细菌中木质素氧化的酶对于木质素生物技术利用为可再生化学产品具有重要意义。两种木质素降解细菌 Ochrobactrum sp. 和 Paenibacillus sp. 的基因组序列均不包含其他细菌中参与木质素降解的 B 型 DyP 过氧化物酶，但含有假定的多铜氧化酶基因。多铜氧化酶 CueO 来自 Ochrobactrum sp. 被表达并重组为重组漆酶样酶，并进行了动力学表征。Ochrobactrum CueO 显示出氧化 β-芳基醚和联苯木质素二聚体模型化合物的活性，生成氧化的二聚体产物，并显示出氧化 Ca-木质素磺酸盐的活性，生成香草酸作为低分子量产物。Ochrobactrum CueO (OcCueO) 的晶体结构已在 1.1 Å分辨率下确定（PDB：6EVG），显示出具有配体 His495、His434 和 Cys490 的四配位单核 I 型铜中心，与大肠杆菌 CueO 和细菌蓝铜蛋白的配体相同，而真菌漆酶酶含有三配位 I 型铜金属中心。三核 2/3 型铜簇被建模到活性部位，显示出与大肠杆菌 CueO 和真菌漆酶相似的结构，以及通向活性部位的三个溶剂通道。对在溶剂通道中发现的氨基酸残基进行了定点突变，表明残基 Asp102、Gly103、Arg221、Arg223 和 Asp462 对催化活性很重要。这项工作鉴定了一种具有木质素氧化活性的新型细菌多铜酶，并暗示细菌漆酶样多铜氧化酶在某些木质素降解细菌中发挥作用。
结构数据可在 PDB 中以访问号 6EVG 获得。
