Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity
Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity
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DOI：
10.1074/jbc.m115.660183
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发表时间：
2015-09-01
影响因子：
4.8
通讯作者：
Eijsink, Vincent G. H.
中科院分区：
文献类型：
--
作者：
Borisova, Anna S.;Isaksen, Trine;Eijsink, Vincent G. H.
最近发现的溶解性多糖单加氧酶（LPMO）进行多糖的氧化裂解，并且对于生物质的有效加工具有重要意义。来自粗糙脉孢菌的NcLPM 0 9 C作用于纤维素和非纤维素β-葡聚糖，包括纤维糊精和木葡聚糖。NcLPMO 9 C的催化结构域的晶体结构揭示了一个扩展的，高极性的底物结合表面非常适合与各种糖底物相互作用。利用NcLPMO 9 C作用于可溶性底物的能力来研究酶-底物相互作用。EPR研究表明，Cu 2 +-中心的环境改变后，基板结合，而等温滴定量热法研究显示，结合亲和力在低微摩尔范围内的聚合物基板，部分是由于存在的碳水化合物结合模块（CBM 1）。重要的是，NcLPMO 9 C的新结构使比较研究成为可能，揭示了LPMO 9 s（C1，C4或两者）的氧化区域选择性与铜配位球的独特结构特征相关。在严格的C1-氧化LPMO 9 s，进入溶剂面临的轴向协调的位置是受限制的保守的酪氨酸残基，而进入这个相同的位置似乎不受限制的C4-氧化LPMO 9 s。已知产生C1-和C4-氧化产物的混合物的LPMO 9显示中间情况。
The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose beta-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+-center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9Cenabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4- oxidized products show an intermediate situation.