Chen Chen, Chen Jinyin, Geng Zhigang, Wang Meixia, Liu Ning, Li Duochuan
Department of Mycology, Shandong Agricultural University, Taian, 271018 Shandong China.
Biotechnol Biofuels. 2018 Jun 5;11:155. doi: 10.1186/s13068-018-1156-2. eCollection 2018.
Polysaccharide monooxygenases (PMOs) of the auxiliary activity 9 (AA9) family have been reported to oxidize C1, C4, and C6 positions in cellulose. However, currently no direct evidence exists that PMOs oxidize C6 positions in cellulose, and molecular mechanism of C1, C4 and C6 oxidation is unclear.
In this study, a PMO gene () belonging to AA9 was isolated from and successfully expressed and correctly processed in . A simple and effective chemical method of using Br to oxidize CtPMO1 reaction products was developed to directly identify C4- and C6-oxidized products by matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF-MS). The PMO (CtPMO1) cleaves phosphoric acid-swollen cellulose (PASC) and celloheptaose, resulting in the formation of oxidized and nonoxidized oligosaccharides. Product identification shows that the enzyme can oxidize C1, C4, and C6 in PASC and cello-oligosaccharides. Mutagenesis of the aromatic residues Tyr27, His64, His157 and residue Tyr206 on the flat surface of CtPMO1 was carried out using site-directed mutagenesis to form the mutated enzymes Y27A, H64A, H157A, and Y206A. It was demonstrated that Y27A retained complete activity of C1, C4, and C6 oxidation on cellulose; Y206A retained partial activity of C1 and C4 oxidation but completely lost activity of C6 oxidation on cellulose; H64A almost completely lost activity of C1, C4, and C6 oxidation on cellulose; and H157A completely lost activity of C1, C4, and C6 oxidation on cellulose.
This finding provides direct and molecular evidence for C1, C4, especially C6 oxidation by lytic polysaccharide monooxygenase. CtPMO1 oxidizes not only C1 and C4 but also C6 positions in cellulose. The aromatic acid residues His64, His157 and residue Tyr206 on CtPMO1 flat surface are involved in activity of C1, C4, C6 oxidation.
据报道，辅助活性9（AA9）家族的多糖单加氧酶（PMO）可氧化纤维素中的C1、C4和C6位。然而，目前尚无直接证据表明PMO能氧化纤维素中的C6位，且C1、C4和C6氧化的分子机制尚不清楚。
在本研究中，从[具体来源]中分离出一个属于AA9的PMO基因（[基因名称]），并在[表达宿主]中成功表达且正确加工。开发了一种简单有效的化学方法，即使用Br氧化CtPMO1反应产物，通过基质辅助激光解吸/电离飞行时间串联质谱（MALDI-TOF-MS）直接鉴定C4和C6氧化产物。PMO（CtPMO1）可切割磷酸膨胀纤维素（PASC）和纤维七糖，产生氧化和未氧化的寡糖。产物鉴定表明，该酶可氧化PASC和纤维寡糖中的C1、C4和C6。利用定点诱变对CtPMO1平面上的芳香族残基Tyr27、His64、His157和残基Tyr206进行诱变，形成突变酶Y27A、H64A、H157A和Y206A。结果表明，Y27A在纤维素上保留了C1、C4和C6氧化的完全活性；Y206A在纤维素上保留了C1和C4氧化的部分活性，但完全丧失了C6氧化活性；H64A在纤维素上几乎完全丧失了C1、C4和C6氧化活性；H157A在纤维素上完全丧失了C1、C4和C6氧化活性。
这一发现为裂解多糖单加氧酶氧化C1、C4尤其是C6提供了直接的分子证据。CtPMO1不仅能氧化纤维素中的C1和C4位，还能氧化C6位。CtPMO1平面上的芳香酸残基His6,4、His157和残基Tyr206参与了C1、C4、C6氧化活性。
