Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis

Summary Coenzyme A ( CoA ) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase ( PanK ). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK , the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase ( KPR ) from the hyperthermophilic archaeon T hermococcus kodakarensis . KPR catalyses the second step in CoA biosynthesis, the reduction of 2‐oxopantoate to pantoate. Gene disruption of TK 1968, whose product was 20–29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK 1968 protein ( Tk ‐ KPR ) displayed reductase activity specific for 2‐oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH ‐dependent enzymes. Tk ‐ KPR activity decreased dramatically in the presence of CoA and KPR activity in cell‐free extracts was also inhibited by CoA . Kinetic studies indicated that CoA inhibits KPR by competing with NADH . Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T . kodakarensis is regulated by feedback inhibition of KPR , providing a feasible regulation mechanism of CoA biosynthesis in archaea.