Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
Host organism: Homo sapiens
Associated disease: hemolytic-uremic syndrome (HUS) [ICD11:
3A21.2 
];
infection due to Escherichia coli [ICD11:
XN6P4 ]
NCBI PubMed ID: 22443398Publication DOI: 10.1021/bi300197hJournal NLM ID: 0370623Publisher: American Chemical Society
Correspondence: Hazel_Holden
biochem.wisc.edu; cleland
biochem.wisc.edu
Institutions: Department of Biochemistry, University of Wisconsin, Madison, WI, USA
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 beta-helix superfamily of proteins. Here we describe a combined structural and functional investigation on 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 beta-sheet. It provides most of the interactions between the protein and GDP-perosamine. The C-terminal domain consists of a left-handed beta-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.
biosynthesis, O-antigen, X-ray, Escherichia coli, gram negative bacteria, binding, Caulobacter crescentus, perosamine N-acetyltransferase, N-acetylperosamine
Structure type: polymer chemical repeating unit
Location inside paper: p.3433
Compound class: O-polysaccharide, O-antigen
Contained glycoepitopes: IEDB_130648,IEDB_136045,IEDB_137473,IEDB_1391961,IEDB_141584,IEDB_142488,IEDB_142489,IEDB_144562,IEDB_146664,IEDB_152214,IEDB_174333,IEDB_885822,IEDB_983931,SB_192,SB_86
Methods: 1H NMR, X-ray, ESI-MS, biochemical methods, HPLC, crystallization
Synthetic data: enzymatic
3D data: 3D data, molecular modeling
Related record ID(s): 28661
NCBI Taxonomy refs (TaxIDs): 83334Reference(s) to other database(s): GTC:G60704LX, GlycomeDB:
3513
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There is only one chemically distinct structure:
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