Dataset Information

Biochemical Characterization of WbkC, an N-Formyltransferase from Brucella melitensis.

ABSTRACT: 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, among others. Indeed, in some species of Brucella, for example, the O-antigen contains 1,2-linked 4-formamido-4,6-dideoxy-?-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. Additionally, 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.

SUBMITTER: Riegert AS

PROVIDER: S-EPMC5543684 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Biochemical Characterization of WbkC, an N-Formyltransferase from Brucella melitensis.

Riegert Alexander S AS Chantigian Daniel P DP Thoden James B JB Tipton Peter A PA Holden Hazel M HM

Biochemistry 20170705 28

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, among 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 GD ...[more]

PMID: 28636341

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Project description:BackgroundAzerbaijan currently ranks thirteenth in global incidence of human brucellosis, with an estimated annual incidence through 2000 at over 50 cases per million. Brucella melitensis has been isolated from patients and is thought to have been acquired through contact with small ruminants or as a foodborne infection. To reduce the burden of human brucellosis, the Azerbaijani government began in 2002, a nationwide vaccination control campaign in small ruminants. There is serological evidence of bovine brucellosis (presumably due to Brucella abortus) in Azerbaijan, but no prevalence estimates were available when this study started in March 2017. The aim of this study was to isolate and identify Brucella spp. from cow milk in the Ganja region, where brucellosis takes a heavy toll on humans and livestock.ResultsBlood and milk samples were collected from cows (n = 1075) in early lactation (up to 90-days) in farms that had a history of previous positive serological results and abortions. Twenty-two out of 57 milk samples collected from seropositive cows, showed growth on Farrell's media, when incubated with 5% CO2. Eight additional milk samples showed growth in the absence of CO2. The classical biotyping classified them as Brucella abortus (22) and Brucella melitensis (8). RT-PCR confirmed that strains belonged to the genus Brucella. MLVA profiles were obtained for DNA extracted from two B. abortus and six B. melitensis strains. While the B. abortus genetic profile was described in the MLVA database, matching the profile of B. abortus strains isolated in East Europe, Central Asia and China, we found a new genotype for the B. melitensis strains isolated in Azerbaijan, clustering with strains belonging to the American clade, rarely identified in the region.ConclusionDespite the implementation of the vaccination program in small ruminants, our results suggest that spill-over events of B. melitensis from small ruminants to cattle have occurred. However, cattle are likely to be primarily infected with B. abortus, which warranted the implementation of a bovine brucellosis program. Such a program started in fall 2017. In the Ganja region, cattle should be considered as a potential source of B. abortus and B. melitensis for humans.

Dataset Information

Biochemical Characterization of WbkC, an N-Formyltransferase from Brucella melitensis.

Publications

Biochemical Characterization of WbkC, an N-Formyltransferase from Brucella melitensis.

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