Identification of DypB from Rhodococcus jostii RHA1 as a Lignin Peroxidase
Identification of DypB from Rhodococcus jostii RHA1 as a Lignin Peroxidase
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DOI：
10.1021/bi101892z
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发表时间：
2011-06-14
期刊：
影响因子：
2.9
通讯作者：
Bugg, Timothy D. H.
中科院分区：
文献类型：
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作者：
Ahmad, Mark;Roberts, Joseph N.;Bugg, Timothy D. H.
Jostii红球菌RHA 1是一株基因组已测序的多氯联苯降解菌，在两种新开发的分光光度法中显示出木质素降解活性。生物信息学分析揭示了两个未注释的过氧化物酶基因存在于R. jostii RHA 1与其他木质素降解微生物中的开放阅读框具有序列相似性。它们是Dyp过氧化物酶家族的成员，基于生物信息学分析，被注释为DypA和DypB。使用比色木质素降解测定的基因缺失突变体的测定揭示了Delta dypB突变体显示出大大降低的木质素降解活性，这与木质素分解中的作用一致。重组DypB蛋白在比色测定中显示出活性，并且使用硫酸盐木质素作为底物显示出Michaelis-Menten动力学行为。使用一系列测定底物，DypB被Mn 2+激活5-23倍，并且在ImM MnCl 2存在下，在24-48小时内观察到重组DypB对麦草木质纤维素的分解。重组DypB与β-芳基醚木质素模型化合物的孵育显示时间依赖性周转，产生香草醛作为产物，表明已经发生了C-alpha-C-beta键裂解。该反应被加入心肌黄酶抑制，与C-C键断裂的自由基机制一致。DypB催化的反应的停流动力学分析显示中间体化合物I（397 nm）与Mn-II（k（obs）= 2.35 s（-1））或β-芳基醚（k（obs）= 3.10 s（-1））之间的反应，在后一种情况下还显示在417 nm处的瞬态，与化合物II中间体一致。这些结果表明，DypB在R. jostii RHA 1能够氧化聚合木质素和木质素模型化合物，并且似乎具有Mn-II和木质素氧化位点。这是重组细菌木质素过氧化物酶的第一个详细表征。
Rhodococcus jostii RHA1, a polychlorinated biphenyl-degrading soil bacterium whose genome has been sequenced, shows lignin degrading activity in two recently developed spectrophotometric assays. Bioinformatic analysis reveals two unannotated peroxidase genes present in the genome of R. jostii RHA1 with sequence similarity to open reading frames in other lignin-degrading microbes. They are members of the Dyp peroxidase family and were annotated as DypA and DypB, on the basis of bioinformatic analysis. Assay of gene deletion mutants using a colorimetric lignin degradation assay reveals that a Delta dypB mutant shows greatly reduced lignin degradation activity, consistent with a role in lignin breakdown. Recombinant DypB protein shows activity in the colorimetric assay and shows Michaelis-Menten kinetic behavior using Kraft lignin as a substrate. DypB is activated by Mn2+ by 5-23-fold using a range of assay substrates, and breakdown of wheat straw lignocellulose by recombinant DypB is observed over 24-48 h in the presence of 1 mM MnCl2. Incubation of recombinant DypB with a beta-aryl ether lignin model compound shows time-dependent turnover, giving vanillin as a product, indicating that C-alpha-C-beta bond cleavage has taken place. This reaction is inhibited by addition of diaphorase, consistent with a radical mechanism for C-C bond cleavage. Stopped-flow kinetic analysis of the DypB-catalyzed reaction shows reaction between the intermediate compound I (397 nm) and either Mn-II (k(obs) = 2.35 s(-1)) or the beta-aryl ether (k(obs) = 3.10 s(-1)), in the latter case also showing a transient at 417 nm, consistent with a compound II intermediate. These results indicate that DypB has a significant role in lignin degradation in R. jostii RHA1, is able to oxidize both polymeric lignin and a lignin model compound, and appears to have both Mn-II and lignin oxidation sites. This is the first detailed characterization of a recombinant bacterial lignin peroxidase.