Molecular structure of an N ‐formyltransferase from P rovidencia alcalifaciens O 30

The existence of N ‐formylated sugars in the O‐antigens of Gram‐negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose‐1‐phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N ‐formyltransferases that utilize N 10 ‐formyltetrahydrofolate as the carbon source. Here we describe the three‐dimensional structure of one of these N ‐formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP‐4‐amino‐4,6‐dideoxyglucose (dTDP‐Qui4N) to dTDP‐4,6‐dideoxy‐4‐formamido‐ d ‐glucose (dTDP‐Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP‐Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed β‐sheet flanked on one side by three α‐helices and on the other side by mostly random coil. This N‐terminal domain is followed by an α‐helix and a β‐hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent‐exposed. Notably, the pyranosyl moiety of dTDP‐Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N ‐formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP‐sugar substrate.