Yuan Shuo-Fu, Wu Tzu-Hui, Lee Hsiao-Lin, Hsieh Han-Yu, Lin Wen-Ling, Yang Barbara, Chang Chih-Kang, Li Qian, Gao Jian, Huang Chun-Hsiang, Ho Meng-Chiao, Guo Rey-Ting, Liang Po-Huang
the Institute of Biochemical Sciences, and.
the Institute of Biotechnology, National Taiwan University, Taipei 10617, Taiwan and.
J Biol Chem. 2015 Feb 27;290(9):5739-48. doi: 10.1074/jbc.M114.604454. Epub 2015 Jan 9.
We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.
我们表达了CtCel5E（一种来自热纤梭菌的双功能纤维素酶/木聚糖酶）的活性形式，进行了生化特性分析，并确定了其无配体和结合配体的晶体结构。从这些结构中可以看出，Asn-93、His-168、His-169、Asn-208、Trp-347和Asn-349与两种配体都存在氢键/疏水相互作用。与来自海栖热袍菌的双功能纤维素酶/甘露聚糖酶同源物TmCel5A的结构相比，CtCel5E中的一个柔性环区域是区分底物的关键。此外，定点诱变数据证实His-168对木聚糖酶活性至关重要，His-169对木聚糖酶活性更为重要，而Asn-93、Asn-208、Tyr-270、Trp-347和Asn-349对两种活性都至关重要。相比之下，F267A提高了酶活性。
