Chitin oligosaccharide binding to the lysin motif of a novel type of chitinase from the multicellular green alga, Volvox carteri
Chitin oligosaccharide binding to the lysin motif of a novel type of chitinase from the multicellular green alga, Volvox carteri
复制标题
几丁质寡糖与来自多细胞绿藻（Volvox carteri）的新型几丁质酶的溶素基序结合
DOI：
10.1007/s11103-016-0549-5
复制
发表时间：
2017
期刊：
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Kitaoku;Y.;Fukamizo;T.;Numata;T. and Ohnuma;T.
关键信息几丁质酶介导的高等植物防御系统已从生理和结构角度进行了深入的研究。然而，绿藻等最原始的植物物种的防御系统尚未被详细阐明。在本研究中，我们解开了红腹蛇几丁质酶N端连接的CBM-50 LysM模块家族的晶体结构，并用核磁共振波谱和等温滴定量热法成功地分析了它与几丁质结合的能力。LysM模块的Trp96与配体的还原末端糖残基发生了CH-π堆积作用。多细胞绿藻Volvox carteri的几丁质酶除含有催化结构域外，还含有属于CBM-50家族的两个N末端裂解蛋白基序(VcLysM1和VcLysM2)。为了研究VcLysM2的结构和功能，我们制备了VcLysM2的重组蛋白。成功地解决了VcLysM2的X-射线晶体结构，发现该蛋白采用了典型的βααβ折叠，属于CBM-50家族成员。对~(13)C和~(15)N标记蛋白质的核磁共振谱进行了分析，以便按顺序完整地指认~1H，~(15)N-HSQC谱的主链共振。甲壳素低聚糖(GlcNAc)n(n = 3-6)的核磁共振滴定实验揭示了VcLysM2的配体结合部位，其中Trp96侧链似乎与配体末端的GlcNAc残基相互作用。然后我们将Trp96突变为丙氨酸(VcLysM2-W96A)，并对突变蛋白进行了鉴定。根据等温滴定量热法发现，(GlcNAc)6对VcLysM2的亲和力(−6.9千卡/摩尔)明显高于(GlcNAc)3(−4.1千卡/摩尔)，而(GlcNAc)6和(GlcNAc)3在VcLysM2-W96A(分别为−5.1千卡/摩尔和−4.0千卡/摩尔)中的亲和力差异不大。这表明VcLysM2的Trp96侧链与(GlcNAc)6的糖残基相互作用，而不是与(GlcNAc)3相互作用。VcLysM2似乎优先与(GlcNAc)n以较长的链结合，在酶靶标的几丁质组分的降解中起主要作用。
Key messageThe chitinase-mediated defense system in higher plants has been intensively studied from physiological and structural viewpoints. However, the defense system in the most primitive plant species, such as green algae, has not yet been elucidated in details. In this study, we solved the crystal structure of a family CBM-50 LysM module attached to the N-terminus of chitinase fromVolvox carteri, and successfully analyzed its chitin-binding ability by NMR spectroscopy and isothermal titration calorimetry. Trp96 of the LysM module appeared to make a CH-π stacking interaction with the reducing end sugar residue of the ligand. We believe the data included in this manuscript provide novel insights into the molecular basis of chitinase-mediated defense system in green algae.AbstractA chitinase from the multicellular green alga,Volvox carteri, contains two N-terminal lysin motifs (VcLysM1 and VcLysM2), that belong to the CBM-50 family, in addition to a catalytic domain. We produced a recombinant protein of VcLysM2 in order to examine its structure and function. The X-ray crystal structure of VcLysM2 was successfully solved at a resolution of 1.2 Å, and revealed that the protein adopts the βααβ fold typical of members belonging to the CBM-50 family. NMR spectra of13C- and15N-labeled proteins were analyzed in order to completely assign the main chain resonances of the1H,15N-HSQC spectrum in a sequential manner. NMR-based titration experiments of chitin oligosaccharides, (GlcNAc)n(n = 3–6), revealed the ligand-binding site of VcLysM2, in which the Trp96 side chain appeared to interact with the terminal GlcNAc residue of the ligand. We then mutated Trp96 to alanine (VcLysM2-W96A), and the mutant protein was characterized. Based on isothermal titration calorimetry, the affinity of (GlcNAc)6toward VcLysM2 (−6.9 kcal/mol) was found to be markedly higher than that of (GlcNAc)3(−4.1 kcal/mol), whereas the difference in affinities between (GlcNAc)6and (GlcNAc)3in VcLysM2-W96A (−5.1 and −4.0 kcal/mol, respectively) was only moderate. This suggests that the Trp96 side chain of VcLysM2 interacts with the sugar residue of (GlcNAc)6not with (GlcNAc)3. VcLysM2 appears to preferentially bind (GlcNAc)nwith longer chains and plays a major role in the degradation of the chitinous components of enzyme targets.