Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara Japan.
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, Japan.
Glycobiology. 2019 Jul 1;29(7):565-575. doi: 10.1093/glycob/cwz024.
Two N-terminal lysin motifs (LysMs) found in a chitinase from the green alga Volvox carteri (VcLysM1 and VcLysM2) were produced, and their structures and chitin-binding properties were characterized. The binding affinities of VcLysM1 toward chitin oligomers determined by isothermal titration calorimetry (ITC) were higher than those of VcLysM2 by 0.8-1.1 kcal/mol of ΔG°. Based on the NMR solution structures of the two LysMs, the differences in binding affinities were found to result from amino acid substitutions at the binding site. The NMR spectrum of a two-domain protein (VcLysM1+2), in which VcLysM1 and VcLysM2 are linked in tandem through a flexible linker, suggested that the individual domains of VcLysM1+2 independently fold and do not interact with each other. ITC analysis of chitin-oligomer binding revealed two different binding sites in VcLysM1+2, showing no cooperativity. The binding affinities of the VcLysM1 domain in VcLysM1+2 were lower than those of VcLysM1 alone, probably due to the flexible linker destabilizing the interaction between the chito-oligosaccahrides and VcLysM1 domain. Overall, two LysMs attached to the chitinase from the primitive plant species, V. carteri, were found to resemble bacterial LysMs reported thus far.
从绿藻衣藻（Volvox carteri）中发现的一种几丁质酶中产生了两个 N 端溶菌酶基序（LysM）（VcLysM1 和 VcLysM2），并对其结构和几丁质结合特性进行了表征。等温滴定微量热法（ITC）测定的 VcLysM1 对几丁质寡聚物的结合亲和力比 VcLysM2 高 0.8-1.1 kcal/mol 的 ΔG°。基于两个 LysM 的 NMR 溶液结构，发现结合亲和力的差异是由于结合部位的氨基酸取代所致。通过柔性接头串联连接的两个结构域蛋白（VcLysM1+2）的 NMR 光谱表明，VcLysM1+2 的各个结构域独立折叠，彼此之间不相互作用。几丁质寡聚物结合的 ITC 分析表明，在 VcLysM1+2 中有两个不同的结合位点，没有协同性。在 VcLysM1+2 中，VcLysM1 结构域的结合亲和力低于单独的 VcLysM1，可能是由于柔性接头使几丁寡糖与 VcLysM1 结构域之间的相互作用不稳定。总的来说，从原始植物物种衣藻（V. carteri）的几丁质酶中发现的两个 LysM 与迄今为止报道的细菌 LysM 相似。
