Structure-function studies of the C3/C5 epimerases and C4 reductases of the Campylobacter jejuni capsular heptose modification pathways.
Structure-function studies of the C3/C5 epimerases and C4 reductases of the Campylobacter jejuni capsular heptose modification pathways.
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DOI：
10.1016/j.jbc.2021.100352
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Creuzenet C
中科院分区：
文献类型：
--
作者：
Barnawi H;Woodward L;Fava N;Roubakha M;Shaw SD;Kubinec C;Naismith JH;Creuzenet C
许多细菌产生以多糖为基础的胶囊，保护它们免受环境的侵害，并在毒力、宿主入侵和其他功能中发挥作用。了解多糖成分是如何合成的，可以为对抗细菌感染提供新的手段。我们之前已经在空肠弯曲杆菌中鉴定了两对参与荚膜糖前体gdp -6-脱氧-d -乙酰庚糖和gdp -6- ome - l -葡萄糖庚糖生物合成的同源酶。然而，这些酶——c3和/或C5外膜酶DdahB和MlghB以及C4还原酶DdahC和mlghc的底物特异性和作用机制尚不清楚。在这里，我们证明了这些酶对庚糖底物具有高度特异性，除了MlghB外，对甘露糖底物的利用效率较低。我们发现DdahB和MlghB具有典型的杯形卷曲褶皱，其具有一系列活动，包括反元化，GDP占据与杯形相似的位置。DdahC和MlghC含有一个罗斯曼折叠，一个催化三联体和一个典型的短链脱水酶还原酶的小c端结构域。将结构信息与位点定向诱变相结合，使我们能够识别每种酶的独特特征，并提供机制见解。在这些外膜酶中，H67、D173、N121、Y134和Y132的突变表明存在替代催化残基。我们发现还原酶可以在没有预先外聚的情况下还原gdp -4-酮-6-脱氧-甘露糖，尽管DdahC更倾向于预外聚的底物，并确定T110和H180对底物特异性和催化效果很重要。这些信息可以用来鉴定用于治疗的抑制剂，或者定制这些酶来合成作为糖生物学工具有用的新糖。
Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6-deoxy-D-altro-heptose and GDP-6-OMe-L-gluco-heptose in Campylobacter jejuni. However, the substrate specificity and mechanism of action of these enzymes—C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC—are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools.
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DOI：
10.1107/s090744491003982x
发表时间：
2011-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Evans PR
通讯作者：
Evans PR
DOI：
10.1107/s0907444911007232
发表时间：
2011-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Krissinel E
通讯作者：
Krissinel E
影响因子：
5.7
作者：
Dong, CJ;Major, LL;Naismith, JH
通讯作者：
Naismith, JH
影响因子：
15
作者：
Major, LL;Wolucka, BA;Naismith, JH
通讯作者：
Naismith, JH
影响因子：
3.6
作者：
Karlyshev, AV;Champion, OL;Szymanski, CM
通讯作者：
Szymanski, CM