Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site.
Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site.
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麦迪霉素在失活位点被多个不同的糖部分失活
DOI：
10.3390/ijms222312636
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发表时间：
2021-11-23
影响因子：
5.6
通讯作者：
Kong JQ
中科院分区：
文献类型：
--
作者：
Lin R;Hong LL;Jiang ZK;Li KM;He WQ;Kong JQ
糖基化失活是大环内酯类抗生素耐药的重要机制之一。积累的证据将糖基化失活归因于大环内酯类化合物失活部位的糖基化修饰。其他糖基化修饰是否会导致大环内酯类化合物失活尚不清楚。在这里，我们证明了不同的糖基化修饰可以导致麦迪霉素失活，麦迪霉素是一种临床和农业上使用的16元大环内酯类抗生素。具体地说，选择了一种放线菌糖基转移酶(GT)OLED，因为它对麦迪霉素的糖多样化能力。OLED能识别UDP-D-葡萄糖、UDP-D-木糖、UDP-半乳糖、UDP-鼠李糖和UDP-N-乙酰氨基葡萄糖胺，生成相应的麦迪霉素2‘-O-糖苷，大部分产物产率较低。因此，进行了OLED的蛋白质工程，以提高其向糖供体的转化率。其中Q327F的转化率最高，是UDP-N-乙酰氨基葡萄糖转化率的7倍。同样，Q327A对UDP-D-木糖的转化率提高了30%。因此，通过蛋白质工程获得了麦迪霉素糖基化的有效生物催化剂。以野生OLED、Q327F和Q327A为生物催化剂，放大制备麦迪霉素2‘-O-吡喃葡萄糖苷、麦迪霉素2’-O-GlcNAc和麦迪霉素2‘-O-木吡喃糖苷。与麦迪霉素相比，这些麦迪霉素2‘-O-糖苷没有显示出抗菌活性。这些证据表明，除糖基化外，其他糖基化方式也能使麦迪霉素失活，为麦迪霉素抗性提供了一种新的失活机制。总体而言，麦迪霉素的糖基化失活与其失活部位的糖基类型无关。
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-N-acetylglucosamine to yield corresponding midecamycin 2′-O-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-N-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′-O-glucopyranoside, midecamycin 2′-O-GlcNAc and midecamycin 2′-O-xylopyranoside. In contrast to midecamycin, these midecamycin 2′-O-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.