Ropoda or Nematoda, although NAMPT and PNC were found in more basal lineages such as the choanoflagellate Monosiga brevicollis and the sea anemone N. vectensis (Figure 1). Such phylogenetic distribution is consistent with a scenario where both genes were present in the Metazoan ancestor and were selectively lost in specific lineages, as evidenced by the different patterns in protostomes. Namely, both genes were found in lophotrochozoans that includes mollusks (Lottia gigantea) and annelids (C. teleta and Helobdella robusta), and the absence of NAMPT was observed in ecdysozoans such as nematodes and arthropods. In deuterostomes, which comprises chordates, hemichordates and echinoderms, both genes were likely present in early lineages, which is supported by the evidence from the extant B. floridae, Saccoglossus kowaleskii and S. purpuratus species, but NAMPT was secondarily lost in the urochordate Ciona intestinalis while PNC was lost in vertebrates (Figure S1). RT-PCR of selected species showed that both NAMPT and PNC genes are expressed in the adult forms of Branchiostoma floridae (Cephalochordata), Strongylocentrotus purpuratus (Echinodermata), Capitella teleta (Annelida) and Nematostella vectensis (Cnidaria) (Figure 1). In addition, available EST (Expressed Sequence Tag) data indicates that NAMPT and PNC genes are also 16985061 co-expressed during developmental stages (Table S2), suggesting a widespread usage of both Nam salvage pathways across Metazoans.Motif conservation in NAMPTs and PNCsWe next used the previously constructed amino acid sequence alignments dataset to search for Title Loaded From File conserved motifs in NAMPT and PNC homologues. In line with the aforementioned results, analyses of NAMPT sequences (Figure 3A) revealed conserved amino acid motifs surrounding catalytic residues [24,25,35?7] Tyr18, Phe193, Asp219, His247, Asp279, Asp313, corresponding to the boxed amino acids in Figure 3. As well, Asp16 and Arg311, Gly353 and Asp354, and Gly384 that bind nicotinamide, ribose or phosphate, respectively, are preserved and the additional NMN interacting residues Arg196 and Gly383 in rat NAMPT [25] are present in all sequences analyzed. The amino acid stretches that represent 23148522 the dimer interface are also conserved in invertebrate NAMPTs (Figure 3A and Figure S2), as previously shown for vertebrates [25]. Similar analyses on PNC homologues showed that, while overall amino acid sequence identity is low (Figure 3B), motifs surrounding metal-binding and catalytic residues (boxed amino acids) show up. Indeed, all PNC sequences have conserved residues that coordinate the metal ion (corresponding to Saccharomyces cerevisiae Asp51, His53 and His94) and the catalytic triad (S. cerevisiae Asp8, Lys122 and Cys167). The characteristic cis-peptide bond that has been identified in available nicotinamidase/pyrazinamidase structures also corresponds to conserved residues present in these species, namely Val-Ala in Pyrococcus horikoshii, S. cerevisiae, Leishmania infantum and C. intestinalis [7,38,39], Ile-Ala in Mycobacterium tuberculosis, Acinetobacter baumanii, H. robusta and B. floridae [40,41], or Val-Leu in Streptococcus pneumoniae [42], and are preceded by a conserved glycine that has a role in catalysis [38,40,41]. Additionally, Title Loaded From File mutations that lead to M. tuberculosis loss of pyrazinamidase activity have defined residues that delineate the active site scaffold [38], corresponding to S. cerevisiae Glu10, Asp12, Phe13, Leu20, His57, Trp91, Gly123, Tyr131, Ser132, V.Ropoda or Nematoda, although NAMPT and PNC were found in more basal lineages such as the choanoflagellate Monosiga brevicollis and the sea anemone N. vectensis (Figure 1). Such phylogenetic distribution is consistent with a scenario where both genes were present in the Metazoan ancestor and were selectively lost in specific lineages, as evidenced by the different patterns in protostomes. Namely, both genes were found in lophotrochozoans that includes mollusks (Lottia gigantea) and annelids (C. teleta and Helobdella robusta), and the absence of NAMPT was observed in ecdysozoans such as nematodes and arthropods. In deuterostomes, which comprises chordates, hemichordates and echinoderms, both genes were likely present in early lineages, which is supported by the evidence from the extant B. floridae, Saccoglossus kowaleskii and S. purpuratus species, but NAMPT was secondarily lost in the urochordate Ciona intestinalis while PNC was lost in vertebrates (Figure S1). RT-PCR of selected species showed that both NAMPT and PNC genes are expressed in the adult forms of Branchiostoma floridae (Cephalochordata), Strongylocentrotus purpuratus (Echinodermata), Capitella teleta (Annelida) and Nematostella vectensis (Cnidaria) (Figure 1). In addition, available EST (Expressed Sequence Tag) data indicates that NAMPT and PNC genes are also 16985061 co-expressed during developmental stages (Table S2), suggesting a widespread usage of both Nam salvage pathways across Metazoans.Motif conservation in NAMPTs and PNCsWe next used the previously constructed amino acid sequence alignments dataset to search for conserved motifs in NAMPT and PNC homologues. In line with the aforementioned results, analyses of NAMPT sequences (Figure 3A) revealed conserved amino acid motifs surrounding catalytic residues [24,25,35?7] Tyr18, Phe193, Asp219, His247, Asp279, Asp313, corresponding to the boxed amino acids in Figure 3. As well, Asp16 and Arg311, Gly353 and Asp354, and Gly384 that bind nicotinamide, ribose or phosphate, respectively, are preserved and the additional NMN interacting residues Arg196 and Gly383 in rat NAMPT [25] are present in all sequences analyzed. The amino acid stretches that represent 23148522 the dimer interface are also conserved in invertebrate NAMPTs (Figure 3A and Figure S2), as previously shown for vertebrates [25]. Similar analyses on PNC homologues showed that, while overall amino acid sequence identity is low (Figure 3B), motifs surrounding metal-binding and catalytic residues (boxed amino acids) show up. Indeed, all PNC sequences have conserved residues that coordinate the metal ion (corresponding to Saccharomyces cerevisiae Asp51, His53 and His94) and the catalytic triad (S. cerevisiae Asp8, Lys122 and Cys167). The characteristic cis-peptide bond that has been identified in available nicotinamidase/pyrazinamidase structures also corresponds to conserved residues present in these species, namely Val-Ala in Pyrococcus horikoshii, S. cerevisiae, Leishmania infantum and C. intestinalis [7,38,39], Ile-Ala in Mycobacterium tuberculosis, Acinetobacter baumanii, H. robusta and B. floridae [40,41], or Val-Leu in Streptococcus pneumoniae [42], and are preceded by a conserved glycine that has a role in catalysis [38,40,41]. Additionally, mutations that lead to M. tuberculosis loss of pyrazinamidase activity have defined residues that delineate the active site scaffold [38], corresponding to S. cerevisiae Glu10, Asp12, Phe13, Leu20, His57, Trp91, Gly123, Tyr131, Ser132, V.
