Unusual α-Carbon Hydroxylation of Proline Promotes Active-Site Maturation

Abstract

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 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.

Role of the Putative Polysaccharide Deacetylase BA1836 from <i>B. anthracis</i> in Spore Development and Germination

“…Additionally, the highly conserved Pro 145 , which we have recently demonstrated to be autocatalytically hydroxylated in the presence of molecular oxygen at the Cα atom to produce 2-hydroxyproline (2-Hyp), is found in motif MT3, in agreement with other well characterized PDAs. The self-hydroxylation of proline at the Cα atom has been shown to significantly enhance enzymatic activity [56]. The pattern of hydrophobic residues in motifs MT4 and MT5 agrees well with the patterns found in other PDAs (Figure 1).…”

Post-Translational Modifications of Protein Backbones: Unique Functions, Mechanisms, and Challenges

“…A case in point for autocatalytic bbPTMs is the C α hydroxylation of an active site proline in a bacterial polysaccharide deacetylase ( Figure 3 a). 18 The additional OH group, installed under aerobic conditions, provides a hydrogen bond to stabilize the tetrahedral intermediate and thus accelerates deacetylase activity. 18 Another series of spontaneous bbPTMs involves asparagine and aspartate residues.…”

“… 18 The additional OH group, installed under aerobic conditions, provides a hydrogen bond to stabilize the tetrahedral intermediate and thus accelerates deacetylase activity. 18 Another series of spontaneous bbPTMs involves asparagine and aspartate residues. These reactions are initiated by nucleophilic attack of a backbone amide nitrogen on the side chain amide (Asn) or, to a lesser extent, acid (Asp).…”

“…(a) α-Hydroxyproline in the active site of Bacillus cereus peptidoglycan N -acetylglucosamine deacetylase (PDB entry 4L1G). 18 (b) Stable succinimide residue in glutaminase from the hyperthermophilic archaeon Pyrococcus horikoshii (PDB entry 1WL8). 21 (c) Isoaspartate-containing hairpin in MurA from Enterobacter cloacae (UDP- N -acetylglucosamine 1-carboxyvinyltransferase, PDB entry 1EJC).…”

Structural and Evolutionary Insights within the Polysaccharide Deacetylase Gene Family of Bacillus anthracis and Bacillus cereus

“…The three-dimensional structures of seven B. anthracis and B. cereus PDAs are now known [ 29 , 31 , 32 , 33 , 34 , 35 , 36 ]. On the structural level, CE4 members contain at least two distinct domains.…”