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Functional and Structural Characterization of Type III Pantothenate Kinase from Pseudomonas aeruginosa
Author(s) -
Leonardi Roberta,
Yun Mi Kyung,
Rock Charles O.,
Jackowski Suzanne
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a470-a
Subject(s) - biochemistry , biosynthesis , kinase , coenzyme a , enzyme , cofactor , biology , escherichia coli , pseudomonas aeruginosa , chemistry , bacteria , reductase , gene , genetics
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 ( Pa CoaA) both functionally and structurally. The crystal structure of the apo form of Pa CoaA 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 Mg 2+ the enzyme absolutely requires a monovalent cation for activity, with NH 4 + 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 Pa CoaA 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 Pa CoaA in a E. coli coaA (Ts) mutant confers resistance to N5‐Pan.