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首页> 外文期刊>Biochemistry >Catalytic Mechanism of Perosamine N-Acetyltransferase Revealed by High-Resolution X-ray Crystallographic Studies and Kinetic Analyses
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Catalytic Mechanism of Perosamine N-Acetyltransferase Revealed by High-Resolution X-ray Crystallographic Studies and Kinetic Analyses

机译：高分辨率X射线晶体学和动力学分析揭示过胺N-乙酰基转移酶的催化机理

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摘要

N-Acetylperosamine is an unusual dideoxysugar found in the O-antigens of some Gram-negative bacteria, including the pathogenic Escherichia coli strain O157:H7. The last step in its biosynthesis is catalyzed by PerB, an N-acetyltransferase belonging to the left-handed β-helix superfamily of proteins. Here we describe a combined structural and functional investigation of PerB from Caulobacter crescentus. For this study, three structures were determined to 1.0 A resolution or better: the enzyme in complex with CoA and GDP-perosamine, the protein with bound CoA and GDP-N-acetylperosamine, and the enzyme containing a tetrahedral transition state mimic bound in the active site. Each subunit of the trimeric enzyme folds into two distinct regions. The N-terminal domain is globular and dominated by a six-stranded mainly parallel β-sheet. It provides most of the interactions between the protein and GDP-perosamine. The C-terminal domain consists of a left-handed β-helix, which has nearly seven turns. This region provides the scaffold for CoA binding. On the basis of these high-resolution structures, site-directed mutant proteins were constructed to test the roles of His 141 and Asp 142 in the catalytic mechanism. Kinetic data and pH—rate profiles are indicative of His 141 serving as a general base. In addition, the backbone amide group of Gly 159 provides an oxyanion hole for stabilization of the tetrahedral transition state. The pH—rate profiles are also consistent with the GDP-linked amino sugar substrate entering the active site in its unprotonated form. Finally, for this investigation, we show that PerB can accept GDP-3-deoxyperosamine as an alternative substrate, thus representing the production of a novel trideoxysugar.
机译：N-乙酰基过胺是一种不常见的双脱氧糖，存在于某些革兰氏阴性细菌的O-抗原中，包括致病性大肠杆菌O157：H7菌株。其生物合成的最后一步是由PerB催化的，PerB是一种N-乙酰基转移酶，属于左旋蛋白质的β-螺旋超家族。在这里，我们描述了来自新月形杆菌的PerB的组合结构和功能研究。在本研究中，确定了三个结构以达到1.0 A或更高的分辨率：与CoA和GDP-perosamine结合的酶，与CoA和GDP-N-乙酰基-过氧化氢结合的蛋白，以及与四面体过渡态模拟物结合的酶活动站点。三聚体酶的每个亚基折叠成两个不同的区域。 N-末端结构域为球形，并以六链主要平行的β-折叠为主。它提供了蛋白质和GDP-过氧化氢之间的大部分相互作用。 C末端结构域由左旋β螺旋组成，该螺旋具有近七匝。该区域提供了用于CoA结合的支架。在这些高分辨率结构的基础上，构建了定点突变蛋白，以测试His 141和Asp 142在催化机理中的作用。动力学数据和pH-速率曲线表明His 141是一般碱。此外，Gly 159的骨架酰胺基团提供了一个氧阴离子孔，以稳定四面体的过渡态。 pH-速率曲线也与以未质子化形式进入活性位点的GDP连接的氨基糖底物一致。最后，对于本研究，我们表明PerB可以接受GDP-3-脱氧过糖胺作为替代底物，从而代表了新型三脱氧糖的生产。

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