Studies on NAD synthetic pathway of hyperthermophile based on genome information

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A gene encoding the L-aspartate oxidase (LAO) homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. We succeeded in expressing the encoding gene in Escherichia coil and purified the product to homogeneity. Characterization of the protein revealed that it was the most thermostable L-aspartate oxidase detected so far. In addition to the oxidase activity, the enzyme catalyzed L-aspartate dehydrogenation in the presence of an artificial electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol and ferricyanide. LAO is known to function as the first enzyme in the de novo NAD biosynthetic pathway in prokaryotes. By a similarity search in public databases, the genes that encode the homologue of all other enzymes involved in the pathway were identified in the P. horikoshi OT-3 genome. A gene encoding nicotinamide mononucleotide adenylytransferase (NMNAT) homologue was overexpressed in E. coli, and the produced enzyme was punfied to homogeneity. Characterization of the enzyme revealed that it is an extremely thermostable NMNAT. The adenylyl group donor specificity was examined by high-performance liquid chromatography. At 70℃, ATP was a prominent donor. However, above 80℃, a relatively small, but significant NMNAT activity was detected when ATP was replaced by ADP or AMP in the reaction mixture. To date, NMNAT that utilizes ADP or AMP as an adenylyl group donor has not been found . The present study provides interesting information in which di-or mono-phosphate nucleotide can be utilized by adenylyltransferase at high temperature. These results suggest that P. horikoshi OT-3 has the de novo NAD biosynthetic pathway under anaerobic conditions. We also discovered a highly stable NAD synthase in Bacillus stearothermophilus. X-ray crystalographic analyses of these enzymes are in progress.