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

Abstract

SummaryCoenzyme 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 Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2-oxopantoate to pantoate. Gene disruption of TK1968, 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 TK1968 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.

Regulation of Coenzyme A Biosynthesis in the Hyperthermophilic Bacterium Thermotoga maritima

“…As there is only one KPR that has been characterized from a hyperthermophile (17), it is difficult to judge, from a biochemical viewpoint, whether the catalytic efficiency of Tm-KPR/KARI is sufficient to conclude that it can act as (27). As T. maritima lacks the classical KPR gene, the relatively higher ratio of KPR activity against KARI activity in Tm-KPR/KARI than that in the E. coli KARI may reflect the fact that the protein does function as a KPR.…”

“…On the other hand, we found that TM0550 was 54.5% identical to the ketol-acid reductoisomerase (KARI) of C. glutamicum, which displays KPR activity (7). As type III PanKs are considered to be unaffected by CoA, we first focused on the KPR reaction, as this is the target of feedback inhibition in archaeal CoA biosynthesis (17). We thus examined whether the KARI homolog TM0550 corresponded to KPR in T. maritima and whether it was inhibited by CoA.…”

“…The presence of PoK and PPS has also been demonstrated in Methanospirillum hungatei (21). The effects of CoA on KPHMT, KPR, PoK, and PPS from T. kodakarensis were examined (16)(17)(18)20) and indicated that KPR is the target of feedback inhibition in this archaeon (17). The crystal structure of KPR complexed with CoA and ketopantoate reveals how CoA inhibits the activity of KPR (22).…”

“…In the hyperthermophilic archaeon Thermococcus kodakarensis, the first four proteins of the CoA biosynthesis pathway and the protein necessary for ␤-alanine synthesis have been identified and characterized (16)(17)(18)(19)(20). Pantoate kinase (PoK) and phosphopantothenate synthetase (PPS) are unique to the archaea and replace PS and PanK in bacteria and eukaryotes in the conversion of pantoate to 4=-phosphopantothenate ( Fig.…”

Metabolism Dealing with Thermal Degradation of NAD + in the Hyperthermophilic Archaeon Thermococcus kodakarensis

“…NAD ϩ is also the starting material for ADP-ribosylation of proteins, a type of posttranslational modification found in eukaryotes and a number of bacteria (2,3). The presence of NAD ϩ -dependent enzymes has been demonstrated in a number of hyperthermophiles (4)(5)(6)(7)(8). However, it is also known that NAD ϩ is readily degraded at high temperature as well as at high pH (9,10).…”