Functional and Structural Characterization of Type III Pantothenate Kinase from Pseudomonas aeruginosa

Abstract

Pantothenate kinase (CoaA) catalyzes the phosphorylation of pantothenate, the first and most regulated step in coenzyme A (CoA) biosynthesis. Regulation of the enzyme through feedback inhibition by CoA or its thioesters controls the intracellular levels of this cofactor in almost all living organisms. Bacteria possess three types of pantothenate kinases that differ in sequence, substrate specificity and regulatory properties. We have characterized the type III CoaA from Pseudomonas aeruginosa (PaCoaA) both functionally and structurally. The crystal structure of the apo form of PaCoaA was determined by the multiwavelength anomalous diffraction method at 2.5 Å resolution. The crystallographic asymmetric unit contains a dimer that shows the ribonuclease H-like fold typical of the sugar kinases/Hsc70/actin superfamily. In addition to Mg2+ the enzyme absolutely requires a monovalent cation for activity, with NH4+ being the strongest activator. In contrast to type I CoaAs, type II and type III CoaAs are refractory to feedback inhibition by CoA or CoA thioesters. Metabolic labeling of P. aeruginosa reveals secretion of pantothenate in the medium indicating that PaCoaA catalyzes the rate limiting step in CoA biosynthesis. N-penthylpantothenamide (N5-Pan) is a pantothenate analog with antimicrobial activity against organisms with either type I or type II CoaAs such as Escherichia coli and Staphylococcus aureus, respectively. Type III CoaAs do not recognize N5-Pan as a substrate either in vitro or in vivo and expression of PaCoaA in a E. coli coaA(Ts) mutant confers resistance to N5-Pan.