Article Figures & Data
Figures
- FIG 1
Salvage pathway for NAD+. The moieties used for the salvage synthesis of NAD+ are represented with different colors (pyridine, yellow; ribose, green; amino group, red). The de novo biosynthesis pathway for NAD+ is also shown with dashed lines. NAD, NAD+; NAM, nicotinamide; NA, nicotinate; ADPR, ADP-ribose; R5P, ribose 5-phosphate; PRPP, phosphoribosyl diphosphate; NaMN, nicotinate mononucleotide; NaAD, nicotinate adenine dinucleotide; QA, quinolinate; NAMase, nicotinamidase; NaMAT, nicotinate mononucleotide adenylyltransferase; NaPRT, nicotinate phosphoribosyltransferase; ADPRP, ADPR pyrophosphatase; RPK, ribose-phosphate pyrophosphokinase; NADS, NAD+ synthase.
- FIG 2
Multiple-sequence alignment of TTHA0328 and other nicotinamidases. The multiple-sequence alignment was performed with ClustalOmega and visualized by ESPript. UniProt accession numbers and organisms from which proteins were sourced are shown on the left side. The position of amino acid residues based on T. thermophilus TTHA0328 product (Q5SLG5 ) is shown. The complete or partially conserved amino acids are colored in red. Triangles and circles indicate the amino acid residues important for the catalytic triad motif and the metal ion-binding motif, respectively.
- FIG 3
Optimal pH and temperature for the activity and thermotolerance of the enzyme. (A) Nicotinamidase activities at different pHs are shown. Triangles, squares, and circles represent activities in different buffers (MES, HEPES, and CHES, respectively). Activities are normalized to the maximum activity at pH 9 and are shown as percentages. (B) Activities at different temperatures are shown. Activities were normalized to the maximum activity at 65°C and are shown as percentage. (C) Residual activities after heat treatment at different temperatures are shown. These activities were normalized by the enzymatic activity without heat treatment (control) and are shown as percentages. Averages and the standard errors from triplicates are shown.
- FIG 4
Phylogenetic tree of nicotinamidases and other cysteine hydrolases. The light green line with an asterisk represents the product of TTHA0328. Lines in light green indicate the proteins that were experimentally characterized as isochorismatase (no. 1 to 4) or nicotinamidase (no. 5 to 10). Blue, orange, red, dark green, and purple lines indicate the protein species belonging to the subfamilies isochorismatase (ISCase), nicotinamidase (NAMase), YcaC-related proteins, NAMase-related proteins, and N-carbamoylsarcosine amidohydrolase (CSHase), respectively, according to the NCBI conserved protein domain family. Black lines indicate the proteins belonging to the cysteine hydrolase superfamily but not to specific subfamilies. 1, P0C6D3 from V. cholerae (ISCase); 2, Q6W4P9 from V. anguillarum (ISCase); 3, Q7DC80 from Pseudomonas aeruginosa (ISCase); 4, P0ADI4 from E. coli (ISCase); 5, A0A0H2UR34 from S. pneumoniae (NAMase); 6, Q8ESQ6 from O. iheyensis (NAMase); 7, D9Q1R5 from A. saccharovorans (NAMase); 8, P53184 from S. cerevisiae (NAMase); 9, A4HRG8 from L. infantum (NAMase); 10, O58727 from P. horikoshii (NAMase); 11, B0VA03 from A. baumannii (NAMase); 12, I6XD65 from M. tuberculosis (NAMase).
- FIG 5
Cell growth of T. thermophilus WT and ΔTTHA0328 deletion strains at different temperatures. The cell growth of the WT strain (A and B) and the ΔTTHA0328 deletion strain (C and D) without nicotinate supplementation (−NA) and with nicotinate supplementation (+NA) are shown. The cultivation temperatures are 70°C (A and C) and 80°C (B and D). The y axes are in the logarithmic scale. Averages and standard errors from biological triplicates are shown.
- FIG 6
Intracellular concentration of NAD+/NADH. The intracellular concentration of NAD+/NADH is shown for the WT strain and the ΔTTHA0328 deletion strain grown at 70°C (A) and 80°C (B). The concentration was determined without nicotinate addition (−NA) and with nicotinate addition (+NA). Averages and standard errors from triplicates are shown. CDW, cell dry weight.
Tables
- TABLE 1
Kinetic parameters of nicotinamidases from different organisms
Organism Km (μM) kcat (s−1) Reference T. thermophilus 17 50 This study S. pneumoniae 2.7 2.1 14 S. cerevisiae 9.6 0.69 43 M. tuberculosis 14 3.1 44 A. saccharovorans 1.8 × 102 77 19 O. iheyensis 2.6 × 102 12 16 - TABLE 2
Bacterial strains, plasmids, and oligonucleotides
Designation Description Source or purpose Strains and mutations Rosetta 2 (DE3) pLysS E. coli used for protein expression Merck Millipore, Germany DH5α E. coli used for cloning TaKaRa, Japan T. thermophilus HB8 Wild type BioResource Center, RIKEN, Japan. ΔTTHA0328 TTHA0328 deletion strain of T. thermophilus This work Plasmids pET11a-TTHA0328 Expression plasmid for nicotinamidase BioResource Center, RIKEN, Japan pUC19-HTK Source of thermostable kanamycin resistance cassette BioResource Center, RIKEN, Japan pUC19-ΔTTHA0328 Plasmid for TTHA0328 deletion, containing HTK gene This work Oligonucleotides U-F 5′-ATTCGAGCTCGGTACCCGGGGGAGACCCGGTCCCCGTA-3′ PCR for upstream region of TTHA0328 U-R 5′-GCCGTCAACGCACCTGGACCATGCCCTC-3′ PCR for upstream region of TTHA0328 D-F 5′-GGTCCAGGTGCGTTGACGGCGGATATGG-3′ PCR for downstream region of TTHA0328 D-R 5′-AAACAAACCGCGTAACCAACATGATTAAGAATTATTAGAGG-3′ PCR for downstream region of TTHA0328 HTK-F 5′-GTTGGTTACGCGGTTTGTTTGAGCACCCCG-3′ PCR for HTK HTK-R 5′-CCTGCAGGTCGACTCTAGAGCCTCGCCGAAGGCGGGAA-3′ PCR for HTK Seq_F 5′-GCGTTGTTGAAGAAGAGCTGGG-3′ PCR for sequencing Seq_R 5′-CTTTAGGGGGAGGACAGGCTTC-3′ PCR for sequencing
Supplemental material
- Supplemental file 1 -
Table S1 (Composition of chemically defined medium for T. thermophilus) and Fig. S1 (Protein purification), S2 (Determination of kinetic parameters of TTHA0328), S3 (Confirmation of the deletion of TTHA0328), and S4 (Nicotinamidase activity in the cell lysate)
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- Supplemental file 1 -
