1] Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland [2].
Nat Commun. 2013;4:2627. doi: 10.1038/ncomms3627.
The initial glycan transfer in asparagine-linked protein glycosylation is catalysed by the integral membrane enzyme oligosaccharyltransferase (OST). Here we study the mechanism of the bacterial PglB protein, a single-subunit OST, using chemically synthesized acceptor peptide analogues. We find that PglB can glycosylate not only asparagine but also glutamine, homoserine and the hydroxamate Asp(NHOH), although at much lower rates. In contrast, N-methylated asparagine or 2,4-diaminobutanoic acid (Dab) are not glycosylated. We find that of the various peptide analogues, only asparagine- or Dab-containing peptides bind tightly to PglB. Glycopeptide products are unable to bind, providing the driving force of product release. We find no suitably positioned residues near the active site of PglB that can activate the acceptor asparagine by deprotonation, making a general base mechanism unlikely and leaving carboxamide twisting as the most likely mechanistic proposal for asparagine activation.
天冬酰胺连接的蛋白糖基化中的初始糖基转移由整合膜酶寡糖基转移酶（OST）催化。在这里，我们使用化学合成的受体肽类似物研究了细菌 PglB 蛋白（一种单一亚基 OST）的机制。我们发现，PglB 不仅可以糖基化天冬酰胺，还可以糖基化谷氨酰胺、高丝氨酸和羟胺基 Asp（NHOH），尽管速度要低得多。相比之下，N-甲基化天冬酰胺或 2,4-二氨基丁酸（Dab）不能被糖基化。我们发现，在各种肽类似物中，只有含有天冬酰胺或 Dab 的肽与 PglB 紧密结合。糖肽产物无法结合，为产物释放提供了驱动力。我们在 PglB 的活性位点附近没有发现合适的位置残基，可以通过去质子化激活受体天冬酰胺，因此不太可能采用一般碱机制，而酰胺扭曲最有可能成为天冬酰胺激活的机制提议。
