The full extent of proline (Pro) hydroxylation has yet to be established, as it is largely unexplored in bacteria. We describe here a so far unknown Pro hydroxylation activity which occurs in active sites of polysaccharide deacetylases (PDAs) from bacterial pathogens, modifying the protein backbone at the C-alpha atom of a Pro residue to produce 2-hydroxyproline (2-Hyp): This process modifies with high specificity- a conserved Pro, shares with the deacetylation reaction the same active site and one catalytic residue, and utilizes molecular oxygen as source for the hydroxyl group oxygen of 2-Hyp. By providing additional hydrogen-bonding capacity, the Pro -> 2-Hyp conversion alters the active site and enhances significantly deacetylase activity, probably by creating a more favorable environment for transition-state stabilization. Our results Classify this process as an active-site "maturation", which is highly atypical in being a protein backbone-modifying activity, rather than a side-chain-modifying one.