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

NAD is an important cofactor for enzymatic oxidation reactions in all living organisms, including (hyper)thermophiles. However, NAD is susceptible to thermal degradation at high temperatures. It can thus be expected that (hyper)thermophiles harbor mechanisms that maintain NAD concentrations and possibly remove and/or reuse undesirable degradation products of NAD Here we confirmed that at 85°C, thermal degradation of NAD results mostly in the generation of nicotinamide and ADP-ribose, the latter known to display toxicity by spontaneously linking to proteins. The hyperthermophilic archaeon possesses a putative ADP-ribose pyrophosphatase (ADPR-PPase) encoded by the TK2284 gene. ADPR-PPase hydrolyzes ADP-ribose to ribose 5-phosphate (R5P) and AMP. The purified recombinant TK2284 protein exhibited activity toward ADP-ribose as well as ADP-glucose. Kinetic analyses revealed a much higher catalytic efficiency toward ADP-ribose, suggesting that ADP-ribose was the physiological substrate. To gain insight into the physiological function of TK2284, a TK2284 gene disruption strain was constructed and examined. Incubation of NAD in the cell extract of the mutant strain at 85°C resulted in higher ADP-ribose accumulation and lower AMP production compared with those in experiments with the host strain cell extract. The mutant strain also exhibited lower cell yield and specific growth rates in a synthetic amino acid medium compared with those of the host strain. The results obtained here suggest that the ADPR-PPase in is responsible for the cleavage of ADP-ribose to R5P and AMP, providing a means to utilize the otherwise dead-end product of NAD breakdown. Hyperthermophilic microorganisms living under high temperature conditions should have mechanisms that deal with the degradation of thermolabile molecules. NAD is an important cofactor for enzymatic oxidation reactions and is susceptible to thermal degradation to ADP-ribose and nicotinamide. Here we show that an ADP-ribose pyrophosphatase homolog from the hyperthermophilic archaeon converts the detrimental ADP-ribose to ribose 5-phosphate and AMP, compounds that can be directed to central carbon metabolism. This physiological role for ADP-ribose pyrophosphatases might be universal in hyperthermophiles, as their homologs are widely distributed among both hyperthermophilic bacteria and archaea.