Bains Rajneesh K, Nasseri Seyed A, Liu Feng, Wardman Jacob F, Rahfeld Peter, Withers Stephen G
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada; Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada; Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
J Biol Chem. 2023 Oct;299(10):105214. doi: 10.1016/j.jbc.2023.105214. Epub 2023 Sep 1.
Sulfation is widespread in nature and plays an important role in modulating biological function. Among the strategies developed by microbes to access sulfated oligosaccharides as a nutrient source is the production of 6-sulfoGlcNAcases to selectively release 6-sulfoGlcNAc from target oligosaccharides. Thus far, all 6-sulfoGlcNAcases identified have belonged to the large GH20 family of β-hexosaminidases. Ηere, we identify and characterize a new, highly specific non-GH20 6-sulfoGlcNAcase from Streptococcus pneumoniae TIGR4, Sp_0475 with a greater than 110,000-fold preference toward N-acetyl-β-D-glucosamine-6-sulfate substrates over the nonsulfated version. Sp_0475 shares distant sequence homology with enzymes of GH20 and with the newly formed GH163 family. However, the sequence similarity between them is sufficiently low that Sp_0475 has been assigned as the founding member of a new glycoside hydrolase family, GH185. By combining results from site-directed mutagenesis with mechanistic studies and bioinformatics we provide insight into the substrate specificity, mechanism, and key active site residues of Sp_0475. Enzymes of the GH185 family follow a substrate-assisted mechanism, consistent with their distant homology to the GH20 family, but the catalytic residues involved are quite different. Taken together, our results highlight in more detail how microbes can degrade sulfated oligosaccharides for nutrients.
硫酸化在自然界中广泛存在，并且在调节生物学功能方面发挥着重要作用。微生物开发的获取硫酸化低聚糖作为营养源的策略之一是产生6-磺基葡糖胺酶，以从目标低聚糖中选择性释放6-磺基葡糖胺。到目前为止，已鉴定出的所有6-磺基葡糖胺酶都属于β-己糖胺酶的大型GH20家族。在此，我们从肺炎链球菌TIGR4中鉴定并表征了一种新的、高度特异性的非GH20 6-磺基葡糖胺酶Sp_0475，它对N-乙酰-β-D-葡糖胺-6-硫酸盐底物的偏好比对非硫酸化版本的偏好高110,000倍以上。Sp_0475与GH20家族的酶以及新形成的GH163家族的酶具有远缘序列同源性。然而，它们之间的序列相似性足够低，以至于Sp_0475被指定为新的糖苷水解酶家族GH185的创始成员。通过将定点诱变的结果与机理研究和生物信息学相结合，我们深入了解了Sp_0475的底物特异性、作用机制和关键活性位点残基。GH185家族的酶遵循底物辅助机制，这与其与GH20家族的远缘同源性一致，但所涉及的催化残基却大不相同。综上所述，我们的结果更详细地突出了微生物如何降解硫酸化低聚糖以获取营养。
