SUPPLEMENTARY INFORMATION A pentose bisphosphate pathway for nucleoside degradation in Archaea Riku Aono 1,, Takaaki Sato 1,, Tadayuki Imanaka, and Haruyuki Atomi 1, * 7 8 9 10 11 1 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 1-810, Japan. Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Noji- Higashi, Kusatsu -877, Japan. JST, CREST, 7, Gobancho, Chiyoda-ku, Tokyo 10-007, Japan. 1 JSPS Research Fellow, Kojimachi, Chiyoda-ku, Tokyo 10-008, Japan. 1 *e-mail: atomi@sbchem.kyoto-u.ac.jp 1 Nature Chemical Biology: doi:10.108/nchembio.178
SUPPLEMENTARY RESULTS SUPPLEMENTARY TABLES 7 8 9 Supplementary Table 1. R1P generation from cytidine by the recombinant TK18 protein coupled with recombinant AMPpase. TK18 AMPpase R1P generation (mm) + +.0 ± 0.0 - + 0.07 ± 0.01 + - 0.08 ± 0.0 - - 0.0 ± 0.0 Reaction conditions and detection methods are described in Online Methods. The data represent the average of three independent experiments and are shown with the SD values. 10 Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Table. R1P generation from R1P by the recombinant TK09 protein. ADP TK09 R1P generation (mm) + +.7 ± 0.10 + - 0.09 ± 0.0 - + 0.0 ± 0.01 Reaction conditions and detection methods are described in Online Methods. The data represent the average of three independent experiments and are shown with the SD values. 7 Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Table. R1P generation in cell-free extracts of wild-type strain. Strain Substrate None KOD1 (wild-type) None + ADP 0.0 ± 0.01 0.0 ± 0.0 None + ATP 0.07 ± 0.0 0.0 ± 0.0 R1P 0.0 ± 0.08 0.0 ± 0.0 R1P + ADP 0.0 ± 0.0.7 ± 0.1 Adenosine 0.0 ± 0.0 0.0 ± 0.0 Adenosine + ADP 0.07 ± 0.0 1. ± 0.19 Guanosine 0.01 ± 0.0 0.01 ± 0.0 Guanosine + ADP 0.0 ± 0.0 1.07 ± 0.08 Uridine 0.0 ± 0.0 0.0 ± 0.01 Uridine + ADP 0.0 ± 0.0 1. ± 0.1 Cytidine 0.00 ± 0.0 0.0 ± 0.01 Cytidine + ADP 0.01 ± 0.0 0.09 ± 0.0 Cytidine + ATP 0.00 ± 0.01 0.09 ± 0.0 AMP 0.0 ± 0.0 0.80 ± 0.1 CMP 0.01 ± 0.0. ± 0. GMP 0.01 ± 0.0 0.11 ± 0.0 UMP 0.0 ± 0.01 0. ± 0.11 Reaction conditions and detection methods are described in Online Methods. The units for all values are mm. The data represent the average of three independent experiments from three independent cultures and are shown with the SD values. 7 Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Table. Response of selected genes towards cytidine. Gene ID Function Mean intensity ratio [log (with cytidine/without cytidine) ± SD] TK0 AMPpase 0.7 ± 0.11 TK018 R1P isomerase 0.17 ± 0.0 TK90 Rubisco 0.1 ± 0.19 TK18 Cytidine kinase 0.00 ± 0.10 TK09 ADP-R1P kinase -0.07 ± 0.1 TK179 Uridine phosphorylase 0.1 ± 0.0 TK18 Purine nucleoside phosphorylase 0.09 ± 0.0 TK189 Purine nucleoside phosphorylase 0.0 ± 0.1 A comparison of transcript levels of selected genes from cells cultivated with or without 10 mm cytidine. The mean intensity ratio is expressed as a log value with the SD values. The data represent the average of four data sets from two independent culture sets and are shown with the SD values. 7 8 Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Table. The effects of phosphate on R1P generation in cell- extracts. Strain Substrate Phosphate Pi None KOD1 KW18 donor (wild-type) (host) R1P ADP + 0.0 ± 0.0.7 ± 0.1.70 ± 0.8 - -0.0 ± 0.0. ± 0..79 ± 0.7 Adenosine ADP + 0.07 ± 0.0 1. ± 0.19 1. ± 0.1-0.0 ± 0.0 0. ± 0.1 0.1 ± 0.19 Guanosine ADP + 0.0 ± 0.0 1.07 ± 0.08 1.0 ± 0.1-0.01 ± 0.0 0. ± 0.11 0.9 ± 0.1 Uridine ADP + 0.0 ± 0.0 1. ± 0.1 1. ± 0.1-0.0 ± 0.0 0. ± 0. 0. ± 0.09 Cytidine ADP + 0.01 ± 0.0 0.09 ± 0.0 0.1 ± 0.0-0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 ATP + 0.00 ± 0.01 0.09 ± 0.0 0.0 ± 0.0-0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Reaction conditions and detection methods are described in Online Methods. The data represent the average of three independent experiments from three independent cultures and are shown with the SD values. 7 8 Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Table. Predicted lengths of the PCR products for confirmation of gene disruption. Target gene Primer sets Strain Host Disruptant TK0 out 1 in 11 - TK18 out 07 in 87 - TK09 out 08 in 88 - TK179 out 0 in 8 - TK18 out 00 in 80 - TK189 out 97 in 77 - The units for all values are bp. Abbreviations: out; the primer sets binding to the outside of the flanking regions, in; the primer sets binding to the inside of the coding region. Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Table 7. Primers used in this study. Primer names TK18f TK18r TK09f TK09r TK0u900f TK0d900r TK18u1000f TK18d1000r TK09u1000f TK09d1000r TK179u1000f TK179d1000r TK18u1000f TK18d1000r TK189u1000f TK189d1000r TK0d1f TK0u1r TK18-87f TK18u1r TK09-87f TK09u1r TK179d1f TK179u1r TK18d1f TK18u1r TK189d1f TK189u1r amppase-out-f amppase-out-r TK18u1100f TK18d1100r TK09u1100f TK09d1100r TK179u1100f TK179d1100r TK18u1100f TK18d1100r TK189u1100f TK189d1100r amppase-in-f amppase-in-r udp-f udp-r mtap1-f mtap1-r mtap-f mtap-r Primer sequences -GGGCATATGCTGGATCTCGTGGTTATCGGTCACGTTTC- -GGGGGATCCTTAGACGGTGATACTCCAGTCCCCCCATTTTT- -AGGCATATGAAGCTGGACGTCATCGGCATCGG- -GGGGGATCCTCAAGTCCTATTGAAGGGCAGATCCAG- -CTGGCTATGATCCCAGAAGACCC- -AGGGCAACGTTATAGCGGCTGC- -AATGATTTACAAGAGTTATCGGAAGCGGAAAAAGA- -AACTGGTGAAATAATGGCCAACGAGTTTGAGA- -GCAAAGCTTAAATCCCTTGCGGGAGATTTT- -CGAACTGGGTGCGGAGTTTCGCGGAGTTCATT- -TACAAGGAAGCCCGCGTTGATGAGCCACTCAA- -ATCATCCTTTGACTTCTTAGACTCTTCTAGCATTT- -TGGACAGGGCGTTTGAGTATTCTTTGGAGATTGAA- -CCTCGCCTCGAAGGTTGCCGTTGAGGCTAGCAT- -GTCAGAAATAGCCTCCTTAAGTCTTCCATAGCCCT- -CTTCCGCAGTATGGCTGTCCACAAGTCCTCCGG- -TCACTTTCTGCATTCTTCTACC- -TCTACCACCCCCTGGGATTG- -TCACCGTCTAACAACAAGATAAAAGAGCAG- -GATACCACCGGAGGCCCTTAGGGGTTCTCCTTAA- -TCAATAGGACTTGAGGCCAACGATTCTCGC- -TTTCACCACCAAAATTGGGAATTGCACTGGGA- -AATGGACGAGAGGCTATTCATTATCAAAACCCT- -GGCAACCACCTTTTTAACTTGATATCATTTACTTT- -TGTTCCTCTTCGTTCTCTCCACTTCTTAACTGT- -TCGCATCACCTAGACAGATAAAAGAAACGTGCCTT- -TTTTTATGGTTTGAATTTTCATTATTTAACACT- -GGTCATCACCGTGTTGATTGGGCGCTTGGATAA- -GCTCGGAACGCTCCCGCTCGGTGTCAT- -GTTGGGCACAAGGACGTTGTTTTCAGA- -CGAGCTTTTACAGGCTCAAAAAGGAAACATACA- -TCCCATCCCCACGCCTTGGGGAGGGCCTTGAGT- -TTCCAGGAGCTCCCTGGTGACCTTTCTGGCGCT- -GGCCTCGTAATGCTTCTGAAAAATATAGGCCCGT- -CCGAACCAGATAGGGGAAGGAGAACTTTGGAA- -TCCGGCCGGTTTAGGCGCTAAAAAGGGTCTAAAGA- -GTTCTCGAAAGCGCCTATGAGGCAATACTCAAAAT- -CGGCGGCTAAGGTCAAGCCAGCAACGTGGGTGGA- -GCTCTCCTCCCACGGAACTCTCATGTTGAGCTCT- -GTAGGACCAGTCACTACCAGGGCCTTAGCCTTT- -GTGAAAGCCAAGATCCGCATACTCGATATGTT- -TCAGATGTTGCCGATTCGCTGGAGAACCATT- -GGGCATATGGTCGAGAAGTTCGTTTCCGCAGAGAGA- -GGGGAATTCTCAACCCCTCATGCCAGGGTGCCATACCCTCTT- -GGGCATATGCCAAGGATAGCGATAATAGGCGGTTCTGGAGTT- -GGGGAATTCCTACTCACCGGTCGCGCCCTTCAGTGCATCCTT- -GGGCATATGCCGAGGATAGGTATTATCGGCGGTTCTGGAGTTT- -GGGGAATTCTCACACAAACATCGTCTTCAGCACATCGGCA- Nature Chemical Biology: doi:10.108/nchembio.178
SUPPLEMENTARY FIGURES Supplementary Fig. 1. Purification of the recombinant TK18 and TK09 proteins. SDS-PAGE analysis was carried out with µg protein per lane, followed by staining with. Coomassie Brilliant Blue. M indicates the marker proteins. 7 8 Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig.. CMP generation from cytidine by the TK18 reaction. (a) Separation of standard compounds using HPLC. The standard compounds used are as follows: mm ATP (black), mm ADP (pink), mm AMP (blue), mm CDP (brown), mm CMP (green) and mm cytidine (navy). (b) Analysis of the products after the TK18 Nature Chemical Biology: doi:10.108/nchembio.178
reaction with mm cytidine and mm ATP as substrates. The lines indicate the products of the reactions with (pink) and without (black) the TK18 protein. In all measurements, HPLC was carried out with a C 18 column COSMOSIL C 18 -PAQ set at 0 C with 0 mm NaH PO (ph.) as the mobile phase. Compounds were detected with a UV detector (A ). Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig.. AMP generation from ADP during the TK09 reaction. mm of ADP (a) and AMP (b) were used as standard references. (c) Analysis of the products after Nature Chemical Biology: doi:10.108/nchembio.178
the TK09 reaction. The three lines indicate the products after reactions with both substrate (R1P) and enzyme (TK09 protein) (black solid line), reactions without R1P (blue dotted line) and reactions without TK09 protein (pink solid line). In all measurements, HPLC was carried out with a C 18 column COSMOSIL C 18 -PAQ set at 0 C with 0 mm NaH PO (ph.) as the mobile phase. Compounds were detected with a UV detector (A ). Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig.. Effect of ph on cytidine kinase activity. Symbols: closed circles; acetate-naoh (ph.0-.), open circles; MES-NaOH (ph.-7.0), closed squares; HEPES- NaOH (ph 7.0-8.0), open squares; Tricine-NaOH (ph 8.0-9.0), closed triangles; CHES- NaOH (ph 9.0-10.0). The data were obtained from single measurements. Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig.. Effect of KCl on cytidine kinase activity. The concentrations of KCl were varied up to 00 mm. The data were obtained from single measurements. Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig.. An Arrhenius plot of cytidine kinase activity at various temperatures. The data were obtained from single measurements. Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig. 7. Specific activities of cytidine kinase with various phosphate donors. The concentration of each phosphate donor was mm along with 10 mm MgCl and 10 mm cytidine. The data represent the average of three independent experiments and are shown with the SD values. Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig. 8. Effect of divalent cations on the activity of cytidine kinase. The concentration of each divalent cations was 0 mm along with 1 mm ATP and 10 mm cytidine. The data represent the average of three independent experiments and are shown with the SD values. N.D.: not detected. Asterisks indicate cations that resulted in precipitation in the reaction mixtures. 7 Nature Chemical Biology: doi:10.108/nchembio.178
7 8 9 10 Supplementary Fig. 9. Kinetic analysis of cytidine kinase. (a) Initial velocities of cytidine kinase were measured with varying concentrations of cytidine (closed circles) or deoxycytidine (open circles) in the presence of a constant concentration of ATP (1 mm) and MgCl (0 mm). (b) Initial velocities of cytidine kinase were measured with varying concentrations of ATP (closed circles) or GTP (open circles) in the presence of a constant concentration of cytidine (10 mm) and MgCl (0 mm). The dotted line is a result of fitting an equation for substrate inhibition (v=v max [S]/(K s +[S]+[S] /K i )) onto the data obtained with GTP. Note that the dotted line does not fit well to the data. The data were obtained from single measurements. 11 Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig. 10. Effect of ph on ADP-R1P kinase activity. Symbols: closed circles; acetate-naoh (ph.0-.), open circles; MES-NaOH (ph.-7.0), closed squares; HEPES-NaOH (ph 7.0-8.0), open squares; Tricine-NaOH (ph 8.0-9.0). The data were obtained from single measurements. Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig. 11. Effect of KCl on ADP-R1P kinase activity. The concentrations of KCl were varied up to 0 mm. The data were obtained from single measurements. Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig. 1. An Arrhenius plot of activity levels of ADP-R1P kinase at various temperatures. The data were obtained from single measurements. Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig. 1. Specific activities of ADP-R1P kinase with various phosphate donors. The concentration of each phosphate donor was mm along with 10 mm MgCl and mm R1P. The data represent the average of three independent experiments and are shown with the SD values. N.D.: not detected. Nature Chemical Biology: doi:10.108/nchembio.178
Supplementary Fig. 1. Effect of divalent cations on the activity of ADP-R1P kinase. The concentration of each divalent cations was 0 mm along with 0 mm ADP and mm R1P. The data represent the average of three independent experiments and are shown with the SD values. N.D.: not detected. Asterisks indicate cations that resulted in precipitation in the reaction mixtures. Nature Chemical Biology: doi:10.108/nchembio.178
7 8 9 10 11 1 Supplementary Fig. 1. Kinetic examination of ADP-R1P kinase. (a) Initial velocities of ADP-R1P kinase with various concentrations of ADP (closed circles) and GDP (open circles) in the presence of a constant concentration of R1P ( mm) with 0 mm MgCl (black) or 0 mm CaCl (gray). Crosses indicate data obtained at ADP concentrations of 0 mm or higher with CaCl, in which we observed a significant amount of white precipitate in the reaction mixtures. Dotted line (1) indicates the curve following Michaelis-Menten kinetics calculated from the complete data set (0-0 mm ADP), whereas dotted line () is based only on the data obtained with 0-1 mm of ADP. (b) Initial velocities of ADP-R1P kinase with varying concentrations of R1P (closed circles) and dr1p (open circles) in the presence of a constant concentration of ADP (0 mm) and MgCl (0 mm). The data were obtained from single measurements. 1 Nature Chemical Biology: doi:10.108/nchembio.178
1 Supplementary Fig. 1. Genotype confirmation of the gene disruption strains by PCR. Individual genes are indicated by their numbers above each panel. M: HindIII-digested λdna marker, H: PCR products using the genomic DNA from the host strain (KW18) as a template, Δ: PCR products using the genomic DNA from the transformants as a template, Nature Chemical Biology: doi:10.108/nchembio.178
out: primer sets that anneal outside of the flanking regions used for homologous recombination, in: primer sets that anneal within the coding region of each gene. Nature Chemical Biology: doi:10.108/nchembio.178