Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop.
The pathogenic bacterium Pseudomonas aeruginosa synthesizes alginate as one of a group of virulence factors that are produced during infections. The enzyme GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis. We show here that penicillic acid is an irreversible inactivator of GDP-mannose dehydrogenase. Inactivation occurs with a rate constant of 0.39+/-0.01 mM(-1) min(-1) at pH 8.0, and does not exhibit saturation behavior. Partial protection from inactivation is afforded by GDP-mannose, but not by the other substrate, NAD+. GMP and NAD+ together provide complete protection against inactivation. Analysis by mass spectrometry confirmed that the enzyme is alkylated at multiple cysteine residues by penicillic acid, including Cys 213, Cys 246, and the active site cysteine, Cys 268. However, the pH dependence of the inactivation rate suggested that alkylation of a single cysteine residue is sufficient to inactivate the enzyme. The C268A mutant protein was also susceptible to inactivation by penicillic acid. The presence of NAD+ and GMP provided partial protection of Cys 246 and Cys 268, and almost complete protection of Cys 213. Cys 213 is located on a helix that forms part of the binding pocket for GDP-mannose, and forms a hydrogen bond with Asn 252. Asn 252 is located on a loop that surrounds GDP-mannose. The C213A mutant enzyme exhibits a Vmax that is 1.8-fold greater than the wild-type enzyme, suggesting that the interaction between Cys 213 and Asn 252 helps to hold the loop in place during catalysis, and that opening the loop to release product is partially rate-limiting. Cys 246 is adjacent to the GDP-mannose binding loop, and its alkylation may also interfere with loop movement.
Kimmel
Jennifer L
JL
Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USA.
Tipton
Peter A
PA
D001665:Binding Sites; D002237:Carbohydrate Dehydrogenases / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000737:chemistry; D003198:Computer Simulation; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010398:Penicillic Acid / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013329:Structure-Activity Relationship
D002237:Carbohydrate Dehydrogenases; C020562:GDPmannose dehydrogenase; D010398:Penicillic Acid