Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria.
Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria.
复制标题
揭示细菌清除唾液酸的一种新的分子机制。
DOI：
10.1074/jbc.ra120.014454
复制
发表时间：
2020-10-02
期刊：
影响因子：
--
通讯作者：
Juge N
中科院分区：
文献类型：
--
作者：
Bell A;Severi E;Lee M;Monaco S;Latousakis D;Angulo J;Thomas GH;Naismith JH;Juge N
人类肠道共生菌瘤胃球菌通过将粘蛋白转化为2，7-脱水-Neu5Ac来清除宿主衍生的N-乙酰神经氨酸(Neu5Ac)。我们之前的研究表明，2，7-脱水-Neu5Ac在被转化回Neu5Ac进行进一步代谢处理之前被转运到Rnnavus atcc 29149中。然而，导致2，7-脱水-Neu5Ac转化为Neu5Ac的分子机制仍然不清楚。利用一维和二维核磁共振技术，我们阐明了氧化还原酶(RgNanOx)通过4-酮-2-脱氧-2，3-脱氢-N-乙酰神经氨酸中间体的形成和NAD+的再生，导致2，7-脱水-Neu5Ac可逆转化为Neu5Ac的多步酶促机理。RgNanOx与NAD+辅因子形成的络合物的晶体结构为罗斯曼折叠的蛋白质二聚体。在RgNanOx结构的指导下，我们通过定点突变鉴定了催化残基。生物信息学分析显示，RgNanOx同源物存在于革兰氏阴性和革兰氏阳性细菌物种中，并与唾液酸转运体共存。电喷雾电离喷雾MS结果表明，大肠杆菌同系物YjhC具有抗2，7-脱氢-Neu5Ac的活性，并能降解2，7-脱氢-Neu5Ac。差示扫描荧光分析证实了YjhC与底物2，7-脱水-Neu5Ac和Neu5Ac以及辅助因子NAD和NADH的结合。最后，利用大肠杆菌突变体和互补生长实验，我们证明了2，7-脱水-Neu5Ac在大肠杆菌中的分解代谢依赖于YjhC和预测的唾液酸转运体YjhB。这些结果揭示了2，7-脱水-Neu5Ac跨菌种分解代谢的分子机制和一种新的唾液酸在大肠杆菌中的转运和分解代谢途径。
The human gut symbiont Ruminococcus gnavus scavenges host-derived N-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro-N-acetylneuraminic acid intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.