Abstract
Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway. The PanK gene from Escherichia coli (coaA) has been previously cloned and the enzyme biochemically characterized; highly related genes exist in other prokaryotes. We isolated a PanK cDNA clone from the eukaryotic fungus Aspergillus nidulans by functional complementation of a temperature-sensitive E. coli PanK mutant. The cDNA clone allowed the isolation of the genomic clone and the characterization of the A. nidulans gene designated panK. The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is expressed constitutively. The amino acid sequence of A. nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and bore little resemblance to its bacterial counterpart, whereas a highly related protein was detected in the genome of Saccharomyces cerevisiae. In contrast to E. coli PanK (bPanK), which is regulated by CoA and to a lesser extent by its thioesters, aPanK activity was selectively and potently inhibited by acetyl-CoA. Acetyl-CoA inhibition of aPanK was competitive with respect to ATP. Thus, the eukaryotic PanK has a distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart.
Highlights
Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway
All 48 colonies grew at the nonpermissive temperature, verifying that complementation was not caused by reversion of the host strain phenotype. These data clearly indicated that the A. nidulans cDNA expressed from phagemid STA1999 encoded the functional equivalent of an active pantothenate kinase
The eukaryotic version of PanK characterized in this report has a primary structure that bears little resemblance to the previously described enzyme expressed in prokaryotic cells
Summary
Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway. The 4Ј-phosphopantetheine portion of CoA is an essential prosthetic group in a number of enzyme systems including the acyl carrier protein components of bacterial and eukaryotic fatty acid synthases [3], citrate lyase [4], ferrichrome synthetase from Aspergillus quadricinctus [5], and malonate decarboxylase of Malonomonas rubra [6]. E. coli produces 15-fold more pantothenate than is required for maintaining the intracellular CoA level [13]. This excess pantothenate is excreted into the medium
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