Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
Host organism: Ovis aries; Bos taurus; Canis lupus familiaris; Sus scrofa
Organ / tissue: brain,
heartAssociated disease: brucellosis [ICD11:
1B95 
, ICD11:
XN22N ];
infection due to Brucella melitensis [ICD11:
XN7ZW ]
NCBI PubMed ID: 636341Publication DOI: 10.1021/acs.biochem.7b00494Journal NLM ID: 0370623Publisher: American Chemical Society
Correspondence: Hazel_Holden
biochem.wisc.edu
Institutions: Department of Biochemistry, University of Wisconsin, Madison, WI, USA, Department of Biochemistry, University of Missouri , Columbia, Missouri 65211, United States
It has become increasingly apparent within the last several years that unusual N-formylated sugars are often found on the O-antigens of such Gram negative pathogenic organisms as Francisella tularensis, Campylobacter jejuni, and Providencia alcalifaciens, amongst others. Indeed, in some species of Brucella, for example, the O-antigen contains 1,2-linked 4-formamido-4,6-dideoxy-α-D-mannosyl groups. These sugars, often referred to as N-formylperosamine, are synthesized in pathways initiating with GDP-mannose. One of the enzymes required for the production of N-formylperosamine, namely WbkC, was first identified in 2000 and was suggested to function as an N-formyltransferase. Its biochemical activity was never experimentally verified, however. Here we describe a combined structural and functional investigation of WbkC from Brucella melitensis. Four high resolution X-ray structures of WbkC were determined in various complexes to address its active site architecture. Unexpectedly, the quaternary structure of WbkC was shown to be different from that previously observed for other sugar N-formyltransferases. In addition, the structures revealed a second binding site for a GDP molecule distinct from that required for GDP-perosamine positioning. In keeping with this additional binding site, kinetic data with the wild type enzyme revealed complex patterns. Removal of GDP binding by mutating Phe 142 to an alanine residue resulted in an enzyme variant displaying normal Michaelis-Menten kinetics. These data suggest that this nucleotide binding pocket plays a role in enzyme regulation. Finally, by using an alternative substrate, we demonstrate that WbkC can be utilized to produce a trideoxysugar not found in nature.
O-antigen, X-ray, Campylobacter jejuni, Brucella melitensis, 6-dideoxy-D-mannose, 4-formamido-4, WbkC
Structure type: homopolymer ; n<100
Location inside paper: p.3657, scheme 1
Compound class: O-antigen
Contained glycoepitopes: IEDB_131172,IEDB_134281,IEDB_1397515,IEDB_2116320,IEDB_434547,IEDB_628715
Methods: X-ray, DNA techniques, kinetics assays, ESI-MS, biochemical methods, HPLC, enzyme assay, crystallization, anion exchange chromatography
Related record ID(s): 12225
NCBI Taxonomy refs (TaxIDs): 29459Reference(s) to other database(s): GTC:G55700WU
Show glycosyltransferases
There is only one chemically distinct structure:
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