Three‐dimensional structure of a sugar N‐formyltransferase from Francisella tularensis

Abstract

N-formylated sugars have been observed on the O-antigens of such pathogenic Gram-negative bacteria as Campylobacter jejuni and Francisella tularensis. Until recently, however, little was known regarding the overall molecular architectures of the N-formyltransferases that are required for the biosynthesis of these unusual sugars. Here we demonstrate that the protein encoded by the wbtj gene from F. tularensis is an N-formyltransferase that functions on dTDP-4-amino-4,6-dideoxy-D-glucose as its substrate. The enzyme, hereafter referred to as WbtJ, demonstrates a strict requirement for N 10 -formyltetrahydrofolate as its carbon source. In addition to the kinetic analysis, the three-dimensional structure of the enzyme was solved in the presence of dTDP-sugar ligands to a nominal resolution of 2.1 Å . Each subunit of the dimeric enzyme is dominated by a "core" domain defined by Met 1 to Ser 185. This core motif harbors the active site residues. Following the core domain, the last 56 residues fold into two a-helices and a b-hairpin motif. The hairpin motif is responsible primarily for the subunit:subunit interface, which is characterized by a rather hydrophobic pocket. From the study presented here, it is now known that WbtJ functions on C-4 0 amino sugars. Another enzyme recently investigated in the laboratory, WlaRD, formylates only C-3 0 amino sugars. Strikingly, the quaternary structures of WbtJ and WlaRD are remarkably different. In addition, there are several significant variations in the side chains that line their active site pockets, which may be important for substrate specificity. Details concerning the kinetic and structural properties of WbtJ are presented.

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

“…8,10 These enzymes function on dTDP-4-amino-4,6-dideoxyglucose (dTDP-Qui4N). Whereas their overall active site architectures are similar to WlaRD, QdtF, and FdtF, their quaternary structures are remarkably different as can be seen in the ribbon representation for VioF in Figure 7c.…”

Molecular architecture of an N-formyltransferase from Salmonella enterica O60

“…Interestingly, they tend to be found in the O-antigens of such pathogenic organisms as Brucella abortus , Providencia alcalifaciens O40, Francisella tularensis , and Campylobacter jejuni (Ovchinnikova et al, 2012; Thoden et al, 2013; Wu and Mackenzie, 1987; Zimmer et al, 2014). …”

“…Since 2013, the three-dimensional structures of four sugar N -formyltransferases have been reported from this laboratory (Genthe et al, 2015; Thoden et al, 2013; Woodford et al, 2015; Zimmer et al, 2014). Two of these enzymes act upon dTDP-3-amino-3,6-dideoxyglucose (dTDP-Qui3N) whereas the other two utilize dTDP-4-amino-4,6-dideoxyglucose as a substrate (dTDP-Qui4N).…”

Enzymes required for the biosynthesis of N-formylated sugars

“…The overall three-dimensional structures of WbtJ and VioF were reported in 2014 and 2015, respectively, and their quaternary structures are decidedly different from that observed for WlaRD as can be seen in Figure 5a [8,24•]. As in WlaRD, the N-terminal domains of WbtJ and VioF harbor the active site region.…”

“…Recent research has demonstrated, for example, that the O-antigens of some Gram-negative bacteria contain quite remarkable formylated dideoxysugars including 3-formamido-3,6-dideoxy-D-glucose (Qui3NFo), 3-formamido-3,6-dideoxy-D-galactose (Fuc3NFo), 4-formamido-4,6-dideoxy-D-glucose (Qui4NFo), and 4-formyl-D-perosamine as depicted in Figure 1b [4]. These unusual sugars have been found on such organisms as Brucella abortus [5], Salmonella enterica O60 [6], Providencia alcalifaciens O40 [7], Francisella tularensis [8], and Campylobacter jejuni [9]. Strikingly, all of the above organisms are extremely pathogenic.…”