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emi4390-sup-0001-si.doc523K

Fig. S1. Complementation tests outline. The aim of this experiment is to exchange the selection marker cassette present in the ACIAD3540::KanR strain by a candidate gene. Primers fwd and rev were used to amplify the target gene. The primers P4 and P5 were extended by the complementary sequences of the integration gene amplification primers. Primers P3 and P6 for the amplification of the flanking regions were chosen to obtain regions of about 300 ± 50 bp. The PCR products (gene and flanking regions R1 and R2) were then combined and amplified to generate a linear fragment containing the target gene flanked by two specific regions of A. baylyi ADP1. Upon recombination, PCR products generated by primers P7 and P8 were sequenced to check the correct locus integration and that no mutations had been introduced during the PCR process. External primers P7 and P8 were chosen to be 800 ± 200 bp upstream and downstream of the cassette. The amplification, transformation and selection protocols were essentially as in de Berardinis and colleagues (2008).

emi4390-sup-0001-si.doc523K

Fig. S2. Growth of A. baylyi ADP1 wild-type and complemented strains. Microwell plates containing triplicate cultures grown aerobically in MAN-U (1 mM) medium at 30°C were monitored every 15 min for 24 h on the Bioscreen (Thermo Scientific Labsystems). ACIAD3540::KanR (referred as to KO3540) is impaired in growth in contrast to the wild-type (WT ACIAD3540) and complemented strains (XCC0279, ACIAD3540, Mvan_5278 and tpucL).

emi4390-sup-0001-si.doc523K

Fig. S3. Steady-state kinetics of urate oxidation in the presence of NADH or NADPH by XCC0279 and the HpxO proteins ACIAD3540 and Mvan_5278. Plots A1–A3: Rates of urate oxidation versus urate concentrations. In plot A1, NADPH is used as the cosubstrate and in plots A2 and A3, NADH is used as the cosubstrate. Plots B1–B3: rates of urate oxidation versus NADH concentrations. Plots C1–C3: rates of urate oxidation versus NADPH concentrations. v is expressed in mole of urate oxidized per minute and per mole of enzyme. In main plots, curves were drawn using the Sigma-Plot program. The Michaelis–Menten equation v = (Vmax S)/(Km + S) applies for plots A1, A2, A3, B3, C1 and C3 and the Hill equation v = (Vmax Sn)/(S50n + Sn) for plots B1, B2 and C2. Insets show the Eadie–Hofstee representation (v versus v/S) of the kinetics. XCC0279 exhibits a Michaelian behaviour toward urate, NADH and NADPH while ACIAD3540 and Mvan_5278 exhibit an Michaelian behaviour only toward urate and a cooperative behaviour toward the reduced nicotinamide nucleotide [as unambiguously shown by the non-linearity of the Eadie-Hofstee plot (v versus v/S)].

emi4390-sup-0001-si.doc523K

Table S1. Oligonucleotide primers used in this study.

emi4390-sup-0001-si.doc523K

Table S2. Steady-state kinetics of urate oxidation by the HpxO proteins ACIAD3540 and Mvan_5278.

emi4390-sup-0001-si.doc523K

Appendix S1. Kinetic characterization of the HpxO proteins ACIAD3540 and Mvan_5278.

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