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, 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. 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.

Investigation of a sugar N‐formyltransferase from the plant pathogen Pantoea ananatis

“…The K M for dTDP‐Qui4N is 0.29 ± 0.05 m M and the k cat is 0.84 ± 0.11 s −1 . The overall catalytic efficiency or k cat / K M for PA1080c is 2.9 × 10 3 (± 400) M −1 s −1 , which is comparable to that observed for other sugar N ‐formyltransferases …”

“…Given our long‐standing interest in unusual sugar biosynthesis, and in particular on N ‐formylated sugars, we utilized a simple bioinformatics analysis to determine whether any strains of P. ananatis contained the genes required for the production of such carbohydrates. Briefly, we performed a BLAST® search to discover gene sequences that were similar to those encoding enzymes previously investigated in the laboratory . The vast majorities of “hits” were annotated as l ‐methionyl‐tRNA N ‐formyltransferases.…”

Misannotations of the genes encoding sugar N‐formyltransferases

“…Whereas N-formylated sugars were first observed in 1985, only recently have reports begun to appear in the literature regarding the structures and functions of the N-formyltransferases that are required for the biosynthesis of Qui4NFo, as well as for 3-formamido-3,6-dideoxy-Dglucose (Qui3NFo), 3-formamido-3,6-dideoxy-D-galactose (Fuc3NFo), and 4-formamido-4,6-dideoxy-D-mannose (N-formylperosamine). [4][5][6][7][8][9][10][11][12][13][14][15] Typically, these sugars, such as Qui3NFo and Qui4NFo, are found on the lipopolysaccharides or lipooligosaccharides of pathogenic Gram-negative bacteria such as Campylobacter jejuni, the organism responsible for gastroenteritis worldwide and Francisella tularensis, the causative agent of rabbit fever. 6,7 N-formylperosamine, on the other hand, has been identified thus far only on the surface homopolysaccharides of Brucella abortus, an intracellular pathogen responsible for chronic bovine disease.…”

The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4‐formamido‐4,6‐dideoxy‐d‐glucose

“…Our results were especially intriguing given that there have been no reports in the literature regarding the existence of N ‐formylated sugars in M. tuberculosis . Typically, these unusual sugars are found on the lipopolysaccharides of pathogenic Gram‐negative bacteria such as Campylobacter jejuni, Francisella tularensis , Providencia alcalifaciens, Salmonella enterica, and Brucella melitensis …”

“…Typically, these unusual sugars are found on the lipopolysaccharides of pathogenic Gram-negative bacteria such as Campylobacter jejuni, Francisella tularensis, Providencia alcalifaciens, Salmonella enterica, and Brucella melitensis. [8][9][10][11][12][13][14][15] Shown in Scheme 1 is the accepted biosynthetic pathway by which N-formylated sugars are produced in bacteria. Rv3404c catalyzes the last step in the pathway.…”