Region of Elongation Factor 1A1 Involved in Substrate Recognition by Legionella pneumophila Glucosyltransferase Lgt1 IDENTIFICATION OF Lgt1 AS A RETAINING GLUCOSYLTRANSFERASE
Region of Elongation Factor 1A1 Involved in Substrate Recognition by Legionella pneumophila Glucosyltransferase Lgt1 IDENTIFICATION OF Lgt1 AS A RETAINING GLUCOSYLTRANSFERASE
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DOI：
10.1074/jbc.m109.008441
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发表时间：
2009-07-24
影响因子：
4.8
通讯作者：
Aktories, Klaus
中科院分区：
文献类型：
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作者：
Belyi, Yury;Stahl, Michael;Aktories, Klaus
Lgt1 是革兰氏阴性菌嗜肺军团菌产生的葡萄糖基转移酶之一。该酶修饰真核延伸因子 1A (eEF1A) 的丝氨酸 53，从而抑制蛋白质合成并导致靶细胞死亡。在这里，我们研究了 eEF1A 区域，该区域对于 Lgt1 的底物识别至关重要。我们报道 eEF1A 的十肽 (50)GKGSFKYAWV(59) 被 Lgt1 有效修饰。该肽覆盖由 eEF1A 的螺旋 A* 和 A' 形成的螺旋-环-螺旋区域的环，并且是螺旋 A' 第一圈的一部分。用丙氨酸取代丝氨酸 53、苯丙氨酸 54、酪氨酸 56 或色氨酸 58 会消除或严重降低糖基化。 Lgt1 修饰的十肽 (50)GKGSFKYAWV(59) 的糖基化率高于从酵母中纯化的全长 eEF1A，表明 eEF1A 的特定构象是 Lgt1 的首选底物。 GenBank (TM) 基于底物十肽搜索相似肽序列，检索到热休克蛋白 70 亚家族 B 抑制因子 1 (Hbs1) 作为 Lgt1 糖基化的靶标。重组 Hbs1 和相应片段 ((303)GKASFAYAWV(312)) 被 Lgt1 糖基化。对葡萄糖基化 eEF1A 衍生的十肽进行 NMR 研究，鉴定出葡萄糖-丝氨酸 53 键的 α-异头结构，并将 Lgt1 表征为保留葡萄糖基转移酶。
Lgt1 is one of the glucosyltransferases produced by the Gram-negative bacterium Legionella pneumophila. This enzyme modifies eukaryotic elongation factor 1A (eEF1A) at serine 53, which leads to inhibition of protein synthesis and death of target cells. Here we studied the region of eEF1A, which is essential for substrate recognition by Lgt1. We report that the decapeptide (50)GKGSFKYAWV(59) of eEF1A is efficiently modified by Lgt1. This peptide covers the loop of the helix-loop-helix region formed by helices A* and A' of eEF1A and is part of the first turn of helix A'. Substitution of either serine 53, phenylalanine 54, tyrosine 56, or tryptophan 58 by alanine abolished or severely decreased glucosylation. Lgt1 modified the decapeptide (50)GKGSFKYAWV(59) with a higher glucosylation rate than full-length eEF1A purified from yeast, suggesting that a specific conformation of eEF1A is the preferred substrate of Lgt1. A GenBank (TM) search on the basis of the substrate decapeptide for similar peptide sequences retrieved heat shock protein 70 subfamily B suppressor 1 (Hbs1) as a target for glucosylation by Lgt1. Recombinant Hbs1 and the corresponding fragment ((303)GKASFAYAWV(312)) were glucosylated by Lgt1. NMR studies with the glucosylated eEF1A-derived decapeptide identified an alpha-anomeric structure of the glucose-serine 53 bond and characterize Lgt1 as a retaining glucosyltransferase.