NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & NHC Key Laboratory of Biosynthesis of Natural Products), Beijing 100050, China.
Int J Mol Sci. 2021 Nov 23;22(23):12636. doi: 10.3390/ijms222312636.
Glycosylation inactivation is one of the important macrolide resistance mechanisms. The accumulated evidences attributed glycosylation inactivation to a glucosylation modification at the inactivation sites of macrolides. Whether other glycosylation modifications lead to macrolides inactivation is unclear. Herein, we demonstrated that varied glycosylation modifications could cause inactivation of midecamycin, a 16-membered macrolide antibiotic used clinically and agriculturally. Specifically, an actinomycetic glycosyltransferase (GT) OleD was selected for its glycodiversification capacity towards midecamycin. OleD was demonstrated to recognize UDP-D-glucose, UDP-D-xylose, UDP-galactose, UDP-rhamnose and UDP--acetylglucosamine to yield corresponding midecamycin 2'--glycosides, most of which displayed low yields. Protein engineering of OleD was thus performed to improve its conversions towards sugar donors. Q327F was the most favorable variant with seven times the conversion enhancement towards UDP--acetylglucosamine. Likewise, Q327A exhibited 30% conversion enhancement towards UDP-D-xylose. Potent biocatalysts for midecamycin glycosylation were thus obtained through protein engineering. Wild OleD, Q327F and Q327A were used as biocatalysts for scale-up preparation of midecamycin 2'--glucopyranoside, midecamycin 2'--GlcNAc and midecamycin 2'--xylopyranoside. In contrast to midecamycin, these midecamycin 2'--glycosides displayed no antimicrobial activities. These evidences suggested that besides glucosylation, other glycosylation patterns also could inactivate midecamycin, providing a new inactivation mechanism for midecamycin resistance. Cumulatively, glycosylation inactivation of midecamycin was independent of the type of attached sugar moieties at its inactivation site.
糖基化失活是大环内酯类抗生素耐药的重要机制之一。大量证据表明，糖基化失活是由于大环内酯类抗生素失活部位的葡糖基化修饰。其他糖基化修饰是否导致大环内酯类抗生素失活尚不清楚。本文研究了不同的糖基化修饰是否会导致米地卡星失活，米地卡星是一种临床上和农业上使用的 16 元大环内酯类抗生素。具体来说，选择了一种放线菌糖基转移酶（GT）OleD，因其对米地卡星具有糖基多样化的能力。实验表明，OleD 能够识别 UDP-D-葡萄糖、UDP-D-木糖、UDP-半乳糖、UDP-鼠李糖和 UDP-N-乙酰葡糖胺，生成相应的米地卡星 2'-糖苷，其中大多数的产率较低。因此，对 OleD 进行了蛋白工程改造，以提高其对糖供体的转化效率。Q327F 是最有利的变体，对 UDP-N-乙酰葡糖胺的转化率提高了 7 倍。同样，Q327A 对 UDP-D-木糖的转化率提高了 30%。通过蛋白工程获得了米地卡星糖基化的有效生物催化剂。野生型 OleD、Q327F 和 Q327A 被用作生物催化剂，用于米地卡星 2'-糖苷、米地卡星 2'-GlcNAc 和米地卡星 2'-木糖苷的规模化制备。与米地卡星不同的是，这些米地卡星 2'-糖苷没有表现出抗菌活性。这些证据表明，除了葡糖基化，其他糖基化模式也能使米地卡星失活，为米地卡星耐药提供了一种新的失活机制。综上所述，米地卡星的糖基化失活与失活部位连接的糖基类型无关。
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