A mouse homologue of the Saccharomyces cerevisiae Pcd1p coenzyme A diphosphatase, NUDT7α, has been expressed as a thioredoxin fusion protein in Escherichia coli. NUDT7α is also a CoA diphosphatase of the nudix hydrolase family, and hydrolyses CoA, CoA esters and oxidized CoA with similar efficiences, yielding 3′,5′-ADP and the corresponding 4′-phosphopantetheine derivative as products. Km and kcat values with CoA were 240μM and 3.8s−1. Activity was optimal at pH8.0 with 5mM Mg2+ or Mn2+ ions, while fluoride was inhibitory with an IC50 value of 20μM. Expression of the Nudt7 gene was highest in liver, intermediate in lung and kidney, and lowest in brain and heart, producing a 1.5kb transcript. A similar pattern of expression was found for the human orthologue, NUDT7. An enzymically inactive splice variant, NUDT7β, which lacks 20 amino acids downstream of the nudix motif, was also found to be expressed in mouse tissues. Transfection of HeLa cells with a vector expressing the Nudt7α gene fused to the C-terminus of red fluorescent protein showed that NUDT7α, like Pcd1p, was a peroxisomal enzyme. The function of the NUDT7 enzyme may be the elimination of oxidized CoA from peroxisomes, or the regulation of CoA and acyl-CoA levels in this organelle in response to metabolic demand.
Abbreviations used: DsRed, Discosoma red fluorescent protein; EST, expressed sequence tag; GFP, green fluorescent protein, IMAGE, integrated molecular analysis of genomes and their expression consortium; IPTG, isopropyl β-d-thiogalactoside; LB, Luria–Bertani broth; Ni-NTA, Ni2+-nitrilotriacetate; ORF, open reading frame; PTS, peroxisomal targeting signal; RT-PCR, reverse-transcriptase PCR; SKL, C-terminal tripeptide PTS1; Trx, thioredoxin.
The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank® Nucleotide Sequence Databases under the accession number AF338424.