Structural and functional characterization of PseC, an aminotransferase involved in the biosynthesis of pseudaminic acid, an essential flagellar modification in Helicobacter pylori
Structural and functional characterization of PseC, an aminotransferase involved in the biosynthesis of pseudaminic acid, an essential flagellar modification in Helicobacter pylori
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DOI：
10.1074/jbc.m512987200
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发表时间：
2006-03-31
影响因子：
4.8
通讯作者：
Young, NM
中科院分区：
文献类型：
--
作者：
Schoenhofen, IC;Lunin, VV;Young, NM
幽门螺杆菌鞭毛蛋白被新型唾液酸样非糖酸、伪胺酸 (Pse) 高度糖基化。糖基化过程对于功能性鞭毛丝的组装和随后的细菌运动至关重要。由于运动性是该病原体和其他重要病原体的关键毒力因素，Pse 生物合成途径为新的治疗靶点提供了潜力。根据最近的 NMR 分析，我们确定 UDP-α-D-GlcNAc 转化为途径中的中心中间体 UDP-4-amino-4,6-dideoxy-beta-L-AltNAc，是通过脱水酶/差向异构酶 PseB (HP0840) 形成 UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose，然后进行氨基转移来进行的通过转氨酶 PseC (HP0366)。 PseC 在幽门螺杆菌 Pse 生物合成途径中的核心作用促使我们确定天然蛋白的晶体结构、其与磷酸吡哆醛单独形成的复合物以及与 UDP-4-氨基-4,6-二脱氧-β-L-AltNAc 产物结合的复合物，后者在酶的活性位点转化为外部醛亚胺形式。在结合位点，AltNAc 糖环采用 C-4(1) 椅子构象，这与溶液中发现的主要 C-1(4) 形式不同。该酶形成同二聚体，其中每个单体都贡献于活性位点，并且这些结构允许鉴定参与氨基转移反应期间β-L-阿拉伯中间体的稳定和可能的催化作用的关键残基。通过定点诱变证实了 Lys(183) 在催化事件中的重要作用。这项工作首次提出了核苷酸-糖转氨酶与其天然配体共结晶，结合该酶的最新功能表征，这些结果将有助于阐明 Pse 生物合成途径中的转氨酶反应机制。
Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-alpha-D-GlcNAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-beta-L-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a C-4(1) chair conformation, which is different from the predominant C-1(4) form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the beta-L-arabino intermediate during the amino transfer reaction. The essential role of Lys(183) in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferaseco-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.