Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD⁺ Salvage through Nicotinamide Deamination

HPLC analysis of the products obtained after reaction with the TK1676 protein. (A) The reaction was carried out using Na and PRPP as the substrates. (B) Enlargement of the area including the NaMN peak in panel A. (C) The reaction was performed using Nm and PRPP as the substrates. (D) Enlargement of the area in which the standard NMN peak can be detected in panel C. Black and gray lines represent the products of reactions with and without TK1676 protein, respectively.

Growth properties of T. kodakarensis host strain KU216 and three gene disruption strains. (A) Growth of the host KU216 strain (circles) and ΔTK1676 mutant (triangles) were examined in a synthetic amino acid medium without Na and Nm (minimal medium, ASW-AA-S⁰-Pyr-Ura-W). (B) Growth of ΔTK0218 mutant in the minimal medium and medium supplemented with Na or Nm are shown. (C) Growth of the ΔTK0218 ΔTK1650 mutant strain in the minimal medium and medium supplemented with Na or Nm. Open, filled, and gray symbols indicate ASW-AA-S⁰-Pyr-Ura-W, ASW-AA-S⁰-Pyr-Ura-W-Na(+), and ASW-AA-S⁰-Pyr-Ura-W-Nm(+) media, respectively. Each value is an average of those from three independent growth experiments. The vertical axis is represented on a logarithmic scale.

Deaminase activity converting Nm to Na in cell extracts. (A) Black and gray lines show reaction products in cell extracts from the KU216 host strain and the ΔTK0218 ΔTK1650 mutant strain, respectively. The experiment was performed three times with cell extracts obtained from three independent cultures for each strain, and a representative result is shown here. (B) The concentrations of Na and Nm were quantified based on peak area in the HPLC analysis. Error bars indicate standard deviations. Filled bars (H) indicate results with the cell extracts of the KU216 host strain, and open bars (Dd) indicate those of the ΔTK0218 ΔTK1650 mutant strain.

Predicted de novo and salvage pathways for NAD⁺ biosynthesis in T. kodakarensis. Thick-lined boxes represent proteins or reactions that have been characterized in T. kodakarensis. Those with stars indicate proteins examined in this study. Dotted boxes show compounds that can be taken up by the cell, based on the results of this study. Other reactions in this figure are based on studies on homologous proteins from other archaea, such as TK0297 homologs from Pyrococcus horikoshii (40), from Sulfolobus tokodaii (41), and from Thermococcus litoralis (42), TK0296 homologs from P. horikoshii (43) and from Pyrococcus furiosus (44), and a TK1798 homolog from Methanocaldococcus jannaschii (38). ADPR-PPase, ADP-ribose pyrophosphatase; DHAP, dihydroxyacetone phosphate; FAD, flavin adenine dinucleotide; NaAD, nicotinic acid adenine dinucleotide; Na, nicotinic acid; NaPRT, nicotinic acid phosphoribosyltransferase; NaMNAT, nicotinic acid mononucleotide adenylyltransferase; Nm, nicotinamide; NmPRT, nicotinamide phosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyltransferase; PRPP, phosphoribosyl pyrophosphate; Qa, quinolinic acid; QaPRT, quinolinic acid phosphoribosyltransferase; R5P, ribose 5-phosphate.