Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform

Abstract

CoA is the major acyl carrier in mammals and a key cofactor in energy metabolism.

Nudt8 is a novel CoA diphosphohydrolase that resides in the mitochondria

“…1A). Indeed, one liter of autoinduced culture typically yielded about 15 mg of Nudt8 compared to the 70-100 mg of Nudt7 obtained per liter of standard E. coli culture [13]. To test the possibility that Nudt8 could hydrolyze CoA species, we expressed and purified the mouse enzyme as a 24 kDa protein with a C-terminal hexahistidine tag (Fig.…”

“…To test the possibility that Nudt8 could hydrolyze CoA species, we expressed and purified the mouse enzyme as a 24 kDa protein with a C-terminal hexahistidine tag (Fig. Analytical gel filtration analysis of recombinant mouse Nudt8 indicated that similar to Nudt7 [13], about 70% of the protein eluted as a monomer (Fig. Compared to recombinant Nudt7, Nudt8 was purified in significantly lower amounts.…”

“…RNA was isolated from flash frozen tissue as previously described [13,30]. Nudt8 mRNA levels were quantified in triplicate by RT-PCR using the Quantitect SYBR Green RT-PCR kit (Qiagen, Germantown, MD, USA) and the primers 5 0 -CAGTTTCCCAGGCGGTAAGT-3 0 and 5 0 -CACGTTG-GCAAGTACTGGGA-3 0 as the forward and reverse primers, respectively.…”

“…HEK293 and HEK293T cells were cultured and transfected as previously described [13]. The amplified product was subcloned into pcDNA3.1(+) (Thermo Fisher Scientific) between EcoRI and XhoI sites.…”

“…In the peroxisomes of liver and kidneys, CoA is hydrolyzed to 3 0 ,5 0 -ADP and (acyl-)phosphopantetheine by two Nudix hydrolases, Nudt7 and Nudt19, respectively [13][14][15][16]. Unlike intact CoA, these smaller degradation products can exit the peroxisomes [17]; thus, the activity of Nudt7 and Nudt19 likely plays an important role in the regulation of the peroxisomal CoA pool and, in turn, CoA-dependent peroxisomal metabolism [12,17,18].…”