Department of Biology, Massachusetts Institute of Technology, 31 Ames St, Cambridge, Massachusetts 02139, United States.
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, Massachusetts 02139, United States.
Biochemistry. 2024 Jan 2;63(1):141-151. doi: 10.1021/acs.biochem.3c00564. Epub 2023 Dec 18.
The genus of Gram-negative bacteria is characterized by the expression of N-linked protein glycosylation (pgl) pathways. As is an emerging human pathogen, a better understanding of the variation of the biosynthetic pathways across the genus is necessary to identify the relationships between protein glycosylation and disease. The pgl pathways of strains have been reported to diverge from other in steps after the biosynthesis of acetylgalactosamine-α1,3-diacetylbacillosamine-α-1-diphosphate undecaprenyl (GalNAc-diNAcBac-PP-Und), which is catalyzed by PglC and PglA, a phosphoglycosyltransferase (PGT) and a glycosyltransferase (GT), respectively. Here we characterize the PglJ GTs from two strains of . Chemical synthesis was employed to access the stereochemically defined glycan donor substrates, uridine diphosphate -acetyl-d-galactosaminuronic acid (UDP-GalNAcA) and uridine diphosphate -acetyl-d-glucosaminuronic acid (UDP-GlcNAcA), to allow biochemical investigation of PglJ. Evidence for the PglJ substrate specificity structural determinants for the C6″ carboxylate-containing sugar was obtained through variant-based biochemical assays. Additionally, characterization of a UDP-sugar dehydrogenase encoded in the pgl operon, which is similar to the WbpO responsible for the oxidization of a UDP-HexNAc to UDP-HexNAcA, supports the availability of a UDP-HexNAcA substrate for a GT that incorporates the modified sugar and provides evidence for the presence of a HexNAcA in the N-linked glycan. Utilizing sequence similarity network (SSN) analysis, we identified conserved sequence motifs among PglJ glycosyltransferases, shedding light on substrate preferences and offering predictive insights into enzyme functions across the genus. These studies now allow detailed characterization of the later steps in the pgl pathway in strains and provide insights into enzyme substrate specificity determinants for glycan assembly enzymes.
革兰氏阴性菌属的特征在于表达 N-连接蛋白糖基化（pgl）途径。由于 是一种新兴的人类病原体，因此有必要更好地了解整个属中生物合成途径的变化，以确定蛋白糖基化与疾病之间的关系。据报道，在乙酰半乳糖胺-α1,3-二乙酰基-β-1,4-二磷酸-β-1-十一碳烯基（GalNAc-diNAcBac-PP-Und）的生物合成之后，pgl 途径在步骤上与其他 菌株有所不同，该步骤由 PglC 和 PglA 催化，分别为磷酸糖基转移酶（PGT）和糖基转移酶（GT）。在这里，我们对来自两种 菌株的 PglJ GT 进行了表征。采用化学合成方法获得了立体定义的糖基供体底物尿苷二磷酸-N-乙酰基-d-半乳糖胺（UDP-GalNAcA）和尿苷二磷酸-N-乙酰基-d-葡萄糖胺（UDP-GlcNAcA），以允许对 PglJ 进行生化研究。通过基于变体的生化测定，获得了 PglJ 底物特异性结构决定因素的证据，这些结构决定因素针对含有 C6″羧酸盐的糖。此外，对 pgl 操纵子中编码的 UDP-糖脱氢酶的表征类似于负责将 UDP-HexNAc 氧化为 UDP-HexNAcA 的 WbpO，这支持了 GT 可获得包含修饰糖的 UDP-HexNAcA 底物，并为 N-连接聚糖中存在 HexNAcA 提供了证据。利用序列相似性网络（SSN）分析，我们鉴定了 PglJ 糖基转移酶之间保守的序列基序，揭示了底物偏好，并为整个 属中糖基化酶的酶功能提供了预测性见解。这些研究现在允许对 菌株中 pgl 途径的后期步骤进行详细表征，并深入了解糖基化酶组装酶的酶底物特异性决定因素。
