4EA9

X-ray structure of GDP-perosamine N-acetyltransferase in complex with transition state analog at 0.9 Angstrom resolution

Summary for 4EA9

• Entry DOI: 10.2210/pdb4ea9/pdb
• Related: 4EA7 4EA8 4EAA 4EAB
• Descriptor: Perosamine N-acetyltransferase, GDP-N-acetylperosamine-coenzyme A, CHLORIDE ION, ... (4 entities in total)
• Functional Keywords: beta helix, acetyltransferase, acetyl coenzyme a, gdp-perosamine, transferase
• Biological source: Caulobacter vibrioides
• Total number of polymer chains: 1
• Total formula weight: 23398.62

Authors

Thoden, J.B.,Reinhardt, L.A.,Cook, P.D.,Menden, P.,Cleland, W.W.,Holden, H.M. (deposition date: 2012-03-22, release date: 2012-04-04, Last modification date: 2023-09-13)

Primary citation

Thoden, J.B.,Reinhardt, L.A.,Cook, P.D.,Menden, P.,Cleland, W.W.,Holden, H.M.
Catalytic Mechanism of Perosamine N-Acetyltransferase Revealed by High-Resolution X-ray Crystallographic Studies and Kinetic Analyses.
Biochemistry, 51:3433-3444, 2012

PubMed Abstract: 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 Å 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.

PubMed: 22443398
DOI: 10.1021/bi300197h

Experimental method

X-RAY DIFFRACTION (0.9 Å)