UBC Theses and Dissertations
Investigating the physiological function of a Staphylococcus aureus Ntn-hydrolase Conroy, Brigid
The N-terminal nucleophile (Ntn)-hydrolase structural superfamily consists of several classes of enzymes, including the penicillin V acylases (PVAs), bile salt hydrolases, and N-acylhomoserine lactone (AHL) acylases. The PVAs use an N-terminal cysteine residue to hydrolyze the amide bond in penicillin V and are most closely related to the bile salt hydrolases. PVAs are encoded by many environmental and pathogenic bacteria and are of great importance in the pharmaceutical industry because the product of penicillin V hydrolysis is used in the production of semi-synthetic antibiotics. Despite their industrial value, the physiological function of the PVAs remains unknown. The opportunistic human pathogen Staphylococcus aureus encodes an Ntn-hydrolase (gene locus SAUSA300_0269). Several potential substrates of SAUSA300_0269 were tested and it was demonstrated to hydrolyze penicillin V but show no activity towards the bile salt glycocholic acid. Based on the observed activity, SAUSA300_0269 was renamed to SaPVA. This enzyme also hydrolyzed several AHLs, which are quorum sensing molecules used by Gram-negative bacteria. SaPVA is the first example of a PVA from a Gram-positive bacterium that cross-reacts with AHLs. The enzyme displayed a preference for unsubstituted AHLs with an acyl chain of six or more carbons. Growth experiments did not support a role for SaPVA in protection of S. aureus against the toxicity of 3-oxo-C12-HSL, an AHL produced by Pseudomonas aeruginosa. Two siderophores used by S. aureus, enterobactin and staphyloferrin A, were also tested as substrates, but SaPVA did not show activity towards either molecule. To obtain further insight into the substrates of SaPVA, the crystal structure was solved to 1.9 Å resolution and compared with those of other characterized PVAs from Gram-positive and Gram-negative bacteria. Similarity in the overall structure and substrate-binding loops of PVAs from Gram-positive bacteria suggest that these enzymes act on similar substrates. Molecular docking was used to predict the binding modes of penicillin V and various AHLs to SaPVA. Docking results provided some clues about the structural features that may be present in physiological substrates of SaPVA, including one or more rings, as well as an aryl group or hydrophobic acyl chain.
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