Department of Advanced Bioscience, Kindai University, 3327-204, Nakamachi, Nara 631-8505, Japan.
Department of Bioscience and Biotechnology, University of the Ryukyus, Okinawa, 903-0213, Japan.
Plant Sci. 2022 Aug;321:111310. doi: 10.1016/j.plantsci.2022.111310. Epub 2022 May 6.
A unique GH18 chitinase containing two N-terminal lysin motifs (PrLysM1 and PrLysM2) was first found in fern, Pteris ryukyuensis (Onaga and Taira, Glycobiology, 18, 414-423, 2008). This type of LysM-chitinase conjugates is not usually found in plants but in fungi. Here, we produced a similar GH18 chitinase with one N-terminal LysM module (EaLysM) from the fern, Equisetum arvense (EaChiA, Inamine et al., Biosci. Biotechnol. Biochem., 79, 1296-1304, 2015), using an Escherichia coli expression system and characterized for its structure and mechanism of action. The crystal structure of EaLysM exhibited an almost identical fold (βααβ) to that of PrLysM2. From isothermal titration calorimetry and nuclear magnetic resonance, the binding mode and affinities of EaLysM for chitooligosaccharides (GlcNAc) (3, 4, 5, and 6) were found to be comparable to those of PrLysM2. The LysM module in EaChiA is likely to bind (GlcNAc) almost independently through CH-π stacking of a Tyr residue with the pyranose ring. The (GlcNAc)-binding mode of LysMs in the LysM-chitinase conjugates from fern plants appears to differ from that of plant LysMs acting in chitin- or Nod-signal perception, in which multiple LysMs cooperatively act on (GlcNAc). Phylogenetic analysis suggested that LysM-GH18 conjugates of fern plants formed a monophyletic group and had been separated earlier than forming the clade of fungal chitinases with LysMs.
首次在蕨类植物肾蕨（Pteris ryukyuensis）中发现了一种含有两个 N 端溶菌酶基序（PrLysM1 和 PrLysM2）的独特 GH18 几丁质酶（Onaga 和 Taira，Glycobiology，18，414-423，2008）。这种类型的 LysM-几丁质酶缀合物通常不在植物中发现，而在真菌中发现。在这里，我们使用大肠杆菌表达系统从蕨类植物问荆（Equisetum arvense）中产生了一种具有一个 N 端 LysM 模块（EaLysM）的类似 GH18 几丁质酶（EaChiA，Inamine 等人，Biosci. Biotechnol. Biochem.，79，1296-1304，2015），并对其结构和作用机制进行了表征。EaLysM 的晶体结构显示出与 PrLysM2 几乎相同的折叠（βααβ）。通过等温滴定量热法和核磁共振，发现 EaLysM 与几丁寡糖（GlcNAc）（3、4、5 和 6）的结合模式和亲和力与 PrLysM2 相当。EaChiA 中的 LysM 模块可能通过 Tyr 残基与吡喃糖环的 CH-π 堆积几乎独立地结合（GlcNAc）。来自蕨类植物的 LysM-几丁质酶缀合物中的 LysM 的（GlcNAc）结合模式似乎与在几丁质或 Nod 信号感知中起作用的植物 LysM 不同，其中多个 LysM 协同作用于（GlcNAc）。系统发育分析表明，蕨类植物的 LysM-GH18 缀合物形成了一个单系群，并且比具有 LysM 的真菌几丁质酶的进化枝更早分离。
