Catalytic Mechanism of Perosamine N-Acetyltransferase Revealed by High-Resolution X-ray Crystallographic Studies and Kinetic Analyses
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- 作者：James B. Thoden ; Laurie A. Reinhardt ; Paul D. Cook ; Patrick Menden ; W. W. Cleland ; Hazel M. Holden
- 刊名：Biochemistry
- 出版年：2012
- 出版时间：April 24, 2012
- 年：2012
- 卷：51
- 期：16
- 页码：3433-3444
- 全文大小：773K
- 年卷期：v.51,no.16(April 24, 2012)
- ISSN：1520-4995
文摘
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 尾-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 脜 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 尾-sheet. It provides most of the interactions between the protein and GDP-perosamine. The C-terminal domain consists of a left-handed 尾-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鈥搑ate 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鈥搑ate 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.