Catalytic mechanism of perosamine N-acetyltransferase revealed by high-resolution X-ray crystallographic studies and kinetic analyses

Biochemistry. 2012 Apr 24;51(16):3433-44. doi: 10.1021/bi300197h. Epub 2012 Apr 10.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetyltransferases / chemistry*
  • Bacterial Proteins / chemistry*
  • Binding Sites
  • Catalysis
  • Catalytic Domain
  • Caulobacter crescentus / enzymology
  • Crystallography, X-Ray
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mannose / analogs & derivatives
  • Mannose / chemistry
  • Mannose / metabolism
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Substrate Specificity


  • Bacterial Proteins
  • 4-amino-4,6-dideoxy-D-mannose
  • Acetyltransferases
  • Mannose