Sáez-Jiménez Verónica, Rencoret Jorge, Rodríguez-Carvajal Miguel Angel, Gutiérrez Ana, Ruiz-Dueñas Francisco Javier, Martínez Angel T
CSIC, Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Department of Biology and Biological Engineering, Chalmers University of Technology, 41296 Gothenburg, Sweden.
Biotechnol Biofuels. 2016 Sep 17;9:198. doi: 10.1186/s13068-016-0615-x. eCollection 2016.
Despite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility.
Two water-soluble sulfonated lignins (from and ) were chemically characterized and used to estimate single electron-transfer rates to the HO-activated VP (native enzyme and mutated variant) transient states (compounds I and II bearing two- and one-electron deficiencies, respectively). When the rate-limiting reduction of compound II was quantified by stopped-flow rapid spectrophotometry, from fourfold (softwood lignin) to over 100-fold (hardwood lignin) lower electron-transfer efficiencies ( values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have ~20-30 % phenolic units, which could be responsible for the observed residual activity. Therefore, methylated (and acetylated) samples were used in new stopped-flow experiments, where negligible electron transfer to the W164S compound II was found. This revealed that the residual reduction of W164S compound II by native lignin was due to its phenolic moiety. Since both native lignins have a relatively similar phenolic moiety, the higher W164S activity on the softwood lignin could be due to easier access of its mono-methoxylated units for direct oxidation at the heme channel in the absence of the catalytic tryptophan. Moreover, the lower electron transfer rates from the derivatized lignosulfonates to native VP suggest that peroxidase attack starts at the phenolic lignin moiety. In agreement with the transient-state kinetic data, very low structural modification of lignin, as revealed by size-exclusion chromatography and two-dimensional nuclear magnetic resonance, was obtained during steady-state treatment (up to 24 h) of native lignosulfonates with the W164S variant compared with native VP and, more importantly, this activity disappeared when nonphenolic lignosulfonates were used.
We demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs (Trp164 of VPL) is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer.
尽管木质素过氧化物酶（VP）和木质素过氧化物酶（LiP）被认为是酶促脱木质素过程中的关键酶，但由于木质素的异质性和低溶解性带来的方法学困难，关于它们与木质素聚合物之间反应速率的信息非常稀少。
对两种水溶性磺化木质素（分别来自[具体来源1]和[具体来源2]）进行了化学表征，并用于估计向HO激活的VP（天然酶和突变变体）瞬态状态（分别带有两个和一个电子缺陷的化合物I和II）的单电子转移速率。当通过停流快速分光光度法定量化合物II的限速还原时，与天然VP相比，表面色氨酸164处的W164S变体对软木木质素的电子转移效率降低了四倍，对硬木木质素的电子转移效率降低了100倍以上。这些木质素磺酸盐含有约20 - 30%的酚类单元，这可能是观察到的残余活性的原因。因此，在新的停流实验中使用了甲基化（和乙酰化）样品，发现向W164S化合物II的电子转移可忽略不计。这表明天然木质素对W164S化合物II的残余还原是由于其酚类部分。由于两种天然木质素具有相对相似的酚类部分，W164S对软木木质素的活性较高可能是因为在没有催化色氨酸的情况下，其单甲氧基化单元更容易进入血红素通道进行直接氧化。此外，衍生化木质素磺酸盐向天然VP的较低电子转移速率表明过氧化物酶攻击始于酚类木质素部分。与瞬态动力学数据一致，通过尺寸排阻色谱和二维核磁共振显示，与天然VP相比，用W164S变体对天然木质素磺酸盐进行稳态处理（长达24小时）期间，木质素的结构修饰非常低，更重要的是，当使用非酚类木质素磺酸盐时，这种活性消失。
我们首次证明，大多数LiP和VP中保守的表面色氨酸（VPL的Trp164）对于氧化木质素聚合物中主要且更难降解的非酚类部分是严格必需的。
