title: Catalytic mechanism of perosamine N-acetyltransferase revealed by high-resolution X-ray crystallographic studies and kinetic analyses.
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.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: James B
authors.initials: JB
authors.lastname: Thoden
authors.forename: Laurie A
authors.initials: LA
authors.lastname: Reinhardt
authors.forename: Paul D
authors.initials: PD
authors.lastname: Cook
authors.forename: Patrick
authors.initials: P
authors.lastname: Menden
authors.forename: W W
authors.initials: WW
authors.lastname: Cleland
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Holden
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D016935:Caulobacter crescentus / Q000201:enzymology; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008358:Mannose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; C057033:4-amino-4,6-dideoxy-D-mannose; D000123:Acetyltransferases; D008358:Mannose