Martini Rachel M, Padhi Chandrashekhar, van der Donk Wilfred A
Department of Biochemistry University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Department of Chemistry and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
J Ind Microbiol Biotechnol. 2024 Dec 31;52. doi: 10.1093/jimb/kuaf028.
Glycocins are a growing family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are O- and/or S-glycosylated. Using a sequence similarity network of putative glycosyltransferases, the thg biosynthetic gene cluster (BGC) was identified in the genome of Thermoanaerobacterium thermosaccharolyticum. Heterologous expression in Escherichia coli showed that the glycosyltransferase (ThgS) encoded in the BGC adds N-acetyl-glucosamine (GlcNAc) to Ser and Cys residues of ThgA. The peptide derived from ThgA, which we name thermoglycocin, was structurally characterized and shown to resemble glycocin F. In addition to two nested disulfide bonds also present in glycocin F, thermoglycocin contains a third disulfide bond creating a C-terminal loop. Unexpectedly, ThgA lacks the common double glycine motif for leader peptide removal by a C39-peptidase. Based on AlphaFold3 modeling, we postulated that cleavage between the leader and core peptide would occur instead at a GK motif, which was experimentally confirmed for an orthologous BGC from Ornithinibacillus bavariensis. Its structurally similar product termed orniglycocin was also produced in E. coli and carries two GlcNAc moieties on two Cys residues. The C39 peptidase domain of the peptidase-containing ATP-binding cassette transporter (PCAT) from this BGC removed the leader peptide after a Gly-Lys motif and the orniglycocin so produced demonstrated antimicrobial activity. This study adds to the small number of characterized glycocins, employs AlphaFold3 to predict the leader peptide cleavage site, and suggests a common naming convention similar to that established for lanthipeptides. One-Sentence Summary: Thermoglycocin from Thermoanaerobacterium thermosaccharolyticum and orniglycocin from Ornithinibacillus bavariensis were produced heterologously in E. coli, shown to contain three disulfide bonds and two GlcNAcylations, and were released by a unique C39 protease that cleaves at a Gly-Lys sequence.
糖霉素是一类不断增加的核糖体合成和翻译后修饰肽（RiPPs），它们进行了O-和/或S-糖基化。利用假定糖基转移酶的序列相似性网络，在嗜热栖热放线菌的基因组中鉴定出了thg生物合成基因簇（BGC）。在大肠杆菌中的异源表达表明，BGC中编码的糖基转移酶（ThgS）将N-乙酰葡糖胺（GlcNAc）添加到ThgA的丝氨酸和半胱氨酸残基上。源自ThgA的肽，我们将其命名为热糖霉素，对其进行了结构表征，结果表明它类似于糖霉素F。除了糖霉素F中也存在的两个嵌套二硫键外，热糖霉素还含有第三个二硫键，形成了一个C端环。出乎意料的是，ThgA缺乏通过C39肽酶去除前导肽的常见双甘氨酸基序。基于AlphaFold3建模，我们推测前导肽和核心肽之间的切割将发生在GK基序处，这一点已通过来自巴伐利亚鸟氨酸芽孢杆菌的直系同源BGC的实验得到证实。其结构相似的产物称为鸟氨酸糖霉素，也在大肠杆菌中产生，并在两个半胱氨酸残基上带有两个GlcNAc部分。来自该BGC的含肽ATP结合盒转运蛋白（PCAT）的C39肽酶结构域在Gly-Lys基序后去除前导肽，如此产生的鸟氨酸糖霉素具有抗菌活性。这项研究增加了已表征糖霉素的数量，利用AlphaFold3预测前导肽切割位点，并提出了一种类似于羊毛硫肽所确立的通用命名法。一句话总结：嗜热栖热放线菌的热糖霉素和巴伐利亚鸟氨酸芽孢杆菌的鸟氨酸糖霉素在大肠杆菌中异源产生，显示含有三个二硫键和两个GlcNAc酰化，并由一种在Gly-Lys序列处切割的独特C39蛋白酶释放。
