Characterization of pyridine nucleotide coenzymes in the hyperthermophilic archaeon Pyrococcus furiosus

Abstract

Pyridine-type nucleotides were identified in cell-free extracts of the hyperthermophilic archaeon Pyrococcus furiosus by their ability to replace authentic nicotinamide adenine dinucleotide (phosphate) [NAD(P)] in assays using pure P. furiosus enzymes. The nucleotides were purified using a combination of ion-exchange and reverse-phase chromatography. They were identified as NAD and NADP by analyses using liquid chromatography-mass spectrometry and high performance liquid chromatography. Their intracellular concentrations were measured in P. furiosus grown using maltose and peptides as the carbon sources. The concentrations decreased during the lag phase but remained constant during the exponential phase at approximately 0.17 and 0.13 mM, respectively. The amount of NAD was significantly lower (more than four-fold lower) than that in mesophilic bacteria, although the NADP concentration was comparable. The internal concentrations of NADH and NADPH in P. furiosus were determined to be 0.14 mM and 0.04 mM, respectively. The overall cellular concentration of NAD(P)(H) in P. furiosus (0.48 mM) is about half the value in the mesophiles. The NAD(H)/NADP(H) ratio in P. furiosus is consistent with the preferred use of NADP by several catabolic enzymes that have been purified from this organism. The mechanisms by which hyperthermophiles stabilize these thermally labile nicotinamide nucleotides are not known.

Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus : Characterization of a Coenzyme A- Dependent NAD(P)H Sulfur Oxidoreductase

“…Purified P. furiosus NSR did not reduce S 0 to H 2 S in detectable amounts in the absence of CoA (or CoA-S-S-CoA), and this cofactor could not be replaced by dephospho-or desulfo-CoA, nor could it be replaced by glutathione or the methanogenic cofactor coenzyme M. The K m value for CoA (10 M) is much lower than the intracellular concentration in P. furiosus (860 M [20]), indicating that NSR would normally be saturated. The affinity of P. furiosus NSR for NADPH (K m , 8.5 mM) is surprisingly low, however, given the intracellular nicotinamide nucleotide concentration in P. furiosus (0.5 mM [34]). This high apparent K m value for NADPH may be related to the proposed mechanism of NADPH oxidation by NSR.…”

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii

“…The reduced nucleotide concentrations have been determined for P. furiosus [15], and while the total concentration of NAD(P)(H) was found to be about half the value seen in the mesophilic Salmonella typhimurium , the pyrococcal NADP(H)/NAD(H) ratio was found to be more than twice that found in S. typhimurium . This result is not surprising, given the presence of several unique NADP + ‐dependent catabolic enzymes present in Pyrococcus [15]. The k cat of 7.2 s −1 determined for the ph CoADR with NADPH as the reducing substrate is similar to the k cat of 27 s −1 observed for the staphylococcal enzyme, although it is lower [12].…”

Investigation of the use of immobilised metal affinity chromatography for the on-line sample clean up and pre-concentration of nucleotides prior to their determination by ion pair liquid chromatography-electrospray mass spectrometry: a pilot study

“…The IMAC-column was conditioned according to the instruction of the supplier of Poros ® 20 MC IMAC material (The Nest Group, Massachusetts, USA) with subsequently: 50 mM EDTA in 1 M NaCl; H 2 O; metal ion solution; H 2 O; 0.1 M NaCl; H 2 O. Because EDTA was not efficient to remove all metal ions from the IMAC-column, a 0.5 M HCl solution was used (The Poros ® stationary phase is stable over the complete pH range [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. The injection of 0.1 M NaCl was omitted as no appreciable differences in chromatographic performance was observed.…”

“…For that purpose a 20 L sample loop was mounted on the 6-port injection valve of the autosampler. The injection Table 2 Summary of experiments in which the influence of the elution plug compositions on both (decoupled) dimensions of nucleotide separation, Fe 3+ -IMAC and the ion pair LC, were tested 4 OAc pH = 6.7 (5) Ø ∼OK/DP 0.2% (w/v) NH 4 OAc pH = 6.7 (6) Ø ∼OK/DP 0.5% (w/v) NH 4 OAc pH = 6.7 (7) Ø Ø/DP 0.1% (w/v) NH 4 loop was subsequently filled with each of the conditioning solutions ( Fig. 6) and the content of the loop was delivered to the IMAC-column at 10 L/min by the auxiliary solvent line.…”

“…In order to understand the role nucleotides play in biochemical processes it was important to develop methods for their separation, quantitation and identification [1][2][3][4][5][6][7][8][9]. Additionally biochemical studies of the metabolism of anti-viral and cytostatic nucleoside-analogues justified the development of new liquid chromatography-mass spectrometry (LC-MS) methods.…”