The nitrate reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofactors. The P. aerophilum nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (V max with nitrate, 1,162 s ؊1 (326 U/mg); V max with chlorate, 1,348 s ؊1 (378 U/mg) [assayed at 75°C]). The K m values for nitrate and chlorate were 58 and 140 M, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was >95°C. When incubated at 100°C, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon.Nitrate serves as electron acceptor to many prokaryotic microbes that thrive under anaerobic conditions. Nitrate respiration occurs via two independent pathways, the denitrification pathway and the ammonification pathway (3). Nitrate is reduced sequentially to dinitrogen gas in the denitrification pathway, while ammonium is the product of the two-step ammonification pathway. The first reaction, in which nitrate is reduced to nitrite via the membrane-bound nitrate reductase, is identical in both pathways (3,21). In general, the dissimilatory nitrate reductase is conserved among bacteria and archaea that have been investigated thus far. The enzyme has been extensively studied in mesophilic nitrate reducing bacteria such as the ammonifier Escherichia coli and the denitrifiers Paracoccus denitrificans, Pseudomonas stuzeri, Pseudomonas denitrificans, and others (5,6,11,12,16). The E. coli NarGHI enzyme is one of the best-characterized enzymes (3). The enzyme complex consists of three subunits (11, 16). The ␣ subunit (NarG) has an M r of 145,000 and contains a molybdopterin cofactor at its active site, where nitrate is reduced to nitrite. The ␤ subunit (NarH) has an M r of 58,000 and is the location of one [3Fe-4S] center and three [4Fe-4S] centers. Both the ␣ and ␤ subunits are attached to the cytoplasmic membrane by the 25,000-Da ␥ subunit (NarI). This polypeptide contains cytochrome b and functions to oxidize the menaquinol or ubiquinol of the quinone pool. Electrons are transferred from the quinol pool via the ␤ subunit to the ␣ subunit active site (3). Recently, nitrate reductases from several archaeal species have been described. While Haloferax volcanii contains a heterotrimeric enzyme complex similar to the bacterial dissimilatory nitrate reductases, the nitrate reductase from Haloferax denitrificans was purified as a heterodimeric enzyme possibly la...

With the development of modern chemistry and biology, non-proteinogenic amino acids (NPAAs) have become a powerful tool for developing peptide-based drug candidates. Drug-like properties of peptidic medicines, due to the smaller size and simpler structure compared to large proteins, can be changed fundamentally by introducing NPAAs in its sequence. While peptides composed of natural amino acids can be used as drug candidates, the majority have shown to be less stable in biological conditions. The impact of NPAA incorporation can be extremely beneficial in improving the stability, potency, permeability, and bioavailability of peptide-based therapies. Conversely, undesired effects such as toxicity or immunogenicity should also be considered. The impact of NPAAs in the development of peptide-based therapeutics is reviewed in this article. Further, numerous examples of peptides containing NPAAs are presented to highlight the ongoing development in peptide-based therapeutics.

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