Characterization of a New Pantothenate Kinase Isoform from Helicobacter pylori

Leisl A Brand,Erick Strauss

doi:10.1074/jbc.c500044200

Abstract

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Two well characterized isoforms of the enzyme are known: a prokaryotic PanK that predominates in eubacteria and a eukaryotic isoform that has primarily been characterized from mammalian and plant sources. Curiously, the genomes of certain pathogenic bacteria, including Helicobacter pylori and Pseudomonas aeruginosa, do not contain a PanK similar to either isoform, although these organisms possess all the other biosynthetic machinery required for CoA production. In this study we cloned, overexpressed and characterized an enzyme from Bacillus subtilis and its homologue from H. pylori and show that they catalyze the ATP-dependent phosphorylation of pantothenate. These enzymes do not share sequence homology with any known PanK, and unlike the bacterial and eukaryotic PanK isoforms their activity is not regulated by either CoA or acetyl-CoA. They also do not accept the pantothenic acid antimetabolite N-pentylpantothenamide as a substrate or are inhibited by it. Taken together, these results point to the identification of a third distinct isoform of PanK that accounts for the only known activity of the enzyme in pathogens such as H. pylori and P. aeruginosa.

Highlights

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms
Recent studies have found that this classification is not unambiguous, as the Staphylococcus aureus enzyme (SaCoaA) has a primary sequence that is closer related to Type II PanKs, and it is not regulated by feedback inhibition [12, 13]
A recent patent application has claimed that the Bacillus subtilis genome contains two separate gene sequences which, when cloned in trans, can each suppress the effects of an Escherichia coli temperature-sensitive mutant defective in EcCoaA activity [16]

Summary

The abbreviations used are

CoA, coenzyme A; PanK or CoaA, pantothenate kinase; EcCoaA, Escherichia coli pantothenate kinase; MmPanK1␤, murine pantothenate kinase; SaCoaA, Staphylococcus aureus pantothenate kinase; BsCoaA, Bacillus subtilis Type I pantothenate kinase; BsCoaX, Bacillus subtilis Type III pantothenate kinase; HpCoaX, Helicobacter pylori Type III pantothenate kinase; PEP, phosphoenolpyruvate; CoaX, Type III pantothenate kinase; AnPanK, Aspergillus nidulans pantothenate kinase; ESI-MS, electrospray ionizationmass spectrometry; contig, group of overlapping clones. Despite our extensive current knowledge of PanK enzymes the identification of this activity remains elusive in a subset of pathogenic bacteria that includes organisms such as Helicobacter pylori and Pseudomonas aeruginosa This fact was highlighted in two recent studies which used a comparative genomics approach to reconstruct the universal CoA biosynthetic pathway in representative organisms of all kingdoms [14, 15]. A recent patent application has claimed that the Bacillus subtilis genome contains two separate gene sequences which, when cloned in trans, can each suppress the effects of an Escherichia coli temperature-sensitive mutant defective in EcCoaA activity [16] While one of these genes encodes the predicted model prokaryotic Type I PanK (BsCoaA), the other shows no homology to any known PanK. Our results show that in comparison to the Type I and Type II PanKs these enzymes exhibit distinctly different characteristics, suggesting that they are the first characterized examples of a third (Type III) PanK isoform

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

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