Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, 940-2188, Japan.
Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, 036-8561, Japan.
Sci Rep. 2020 Nov 26;10(1):20614. doi: 10.1038/s41598-020-77462-8.
Sphingobium sp. strain SYK-6 is an alphaproteobacterial degrader of lignin-derived aromatic compounds, which can degrade all the stereoisomers of β-aryl ether-type compounds. SYK-6 cells convert four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGE) into two enantiomers of α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) through GGE α-carbon atom oxidation by stereoselective Cα-dehydrogenases encoded by ligD, ligL, and ligN. The ether linkages of the resulting MPHPV enantiomers are cleaved by stereoselective glutathione (GSH) S-transferases (GSTs) encoded by ligF, ligE, and ligP, generating (βR/βS)-α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. To date, it has been shown that the gene products of ligG and SLG_04120 (ligQ), both encoding GST, catalyze GSH removal from (βR/βS)-GS-HPV, forming achiral β-hydroxypropiovanillone. In this study, we verified the enzyme properties of LigG and LigQ and elucidated their roles in β-aryl ether catabolism. Purified LigG showed an approximately 300-fold higher specific activity for (βR)-GS-HPV than that for (βS)-GS-HPV, whereas purified LigQ showed an approximately six-fold higher specific activity for (βS)-GS-HPV than that for (βR)-GS-HPV. Analyses of mutants of ligG, ligQ, and both genes revealed that SYK-6 converted (βR)-GS-HPV using both LigG and LigQ, whereas only LigQ was involved in converting (βS)-GS-HPV. Furthermore, the disruption of both ligG and ligQ was observed to lead to the loss of the capability of SYK-6 to convert MPHPV. This suggests that GSH removal from GS-HPV catalyzed by LigG and LigQ, is essential for cellular GSH recycling during β-aryl ether catabolism.
鞘氨醇单胞菌属菌株 SYK-6 是一种能够降解木质素衍生芳香族化合物的α变形菌，它可以降解所有β-芳基醚型化合物的立体异构体。SYK-6 细胞通过立体选择性 Cα-脱氢酶 ligD、ligL 和 ligN 对愈创木基甘油-β-愈创木基醚（GGE）的α-碳原子氧化，将 GGE 的四个立体异构体转化为两种对映体的α-（2-甲氧基苯氧基）-β-羟基丙酰香草酮（MPHPV）。所得 MPHPV 对映体的醚键通过立体选择性谷胱甘肽 S-转移酶（GSTs） ligF、ligE 和 ligP 切割，生成（βR/βS）-α-谷胱甘肽-β-羟基丙酰香草酮（GS-HPV）和愈创木酚。迄今为止，已经表明 ligG 和 SLG_04120（ligQ）的基因产物都编码 GST，它们催化从（βR/βS）-GS-HPV 中去除 GSH，形成非手性的β-羟基丙酰香草酮。在本研究中，我们验证了 LigG 和 LigQ 的酶性质，并阐明了它们在β-芳基醚代谢中的作用。纯化的 LigG 对（βR）-GS-HPV 的比活性约为（βS）-GS-HPV 的 300 倍，而纯化的 LigQ 对（βS）-GS-HPV 的比活性约为（βR）-GS-HPV 的 6 倍。对 ligG、ligQ 和两个基因的突变体进行分析表明，SYK-6 使用 LigG 和 LigQ 转化（βR）-GS-HPV，而只有 LigQ 参与转化（βS）-GS-HPV。此外，观察到 ligG 和 ligQ 的缺失会导致 SYK-6 丧失转化 MPHPV 的能力。这表明 LigG 和 LigQ 催化的 GS-HPV 中的 GSH 去除对于β-芳基醚代谢过程中的细胞 GSH 循环回收是必不可少的。
