Fig. 4. Putative catalytic mechanism of ArnT.

(A) Schematic representation of substrate-binding–induced conformational changes and catalytic cycle of ArnT_Cm. The boundaries of cavity 1 are shown in green, and the loop (PL4) that rearranges upon UndP binding is in red. (B) Structural perspective of the active site of ArnT_Cm. A putative position for the aminoarabinose sugar determined by docking is shown. The conserved D55 and D158 are located at the interface between the binding site for UndP-L-Ara4N and cavity 1, and have a Cγ-Cγ distance of 7.1 Å. P1 (magenta) is the phosphate of experimentally determined UndP, whereas P2 (heteroatom) is the phosphate from the modeled L-Ara4N-phosphate. (C) Putative catalytic mechanism in which two of the oxygen atoms of the acceptor phosphate are coordinated by D55 and D158, which leaves the third with a net negative charge, primed for a direct nucleophilic attack on the arabinose ring. This mechanism is consistent with inversion of the glycosidic bond, as reported for L-Ara4N attachment to lipid A (23). A precedent for a phosphate acting as a nucleophile exists (24). R58 and K203 appear to act similarly as the catalytic Mg²⁺, in the case of PglB, that localizes the charge of an aspartate and a glutamate, which in turn coordinate the nucleophile acceptor amide (19, 25). (D) Function of putative catalytic aspartates D32 and D136 and positively charged residues R35 and K180 in ArnT_Se tested utilizing the PMXB growth assay (13). Data presented are means + SD. N is shown for each data column.