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Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis
Author(s) -
Tomita Hiroya,
Imanaka Tadayuki,
Atomi Haruyuki
Publication year - 2013
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12363
Subject(s) - biosynthesis , reductase , biochemistry , biology , coenzyme a , enzyme , archaea , cofactor , bacteria , transferase , mevalonic acid , gene , genetics
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.