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Microbial urate catabolism: characterization of HpyO, a non-homologous isofunctional isoform
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of the flavoprotein urate hydroxylase HpxO.
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Magalie Michiel1,2,3,4, Nadia Perchat1,2,3, Alain Perret1,2,3, Sabine Tricot1,2,3, Aude Papeil1,2,3,
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Marielle Besnard1,2,3, Véronique de Berardinis1,2,3, Marcel Salanoubat1,2,3, and Cécile Fischer1,2,3, #
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From the Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA), DSV, Institut de
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Génomique (IG)
Université d’Evry Val d’Essonne (UEVE)
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CNRS UMR 8030, 2 rue Gaston Crémieux, F-91057 Evry Cedex, France
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Université de Cergy-Pontoise, Laboratoire SATIE, ENSC, CNRS UMR 8029, 5 mail Gay Lussac, F-
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95031 Neuville sur Oise cedex, France
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UMR8030, 2 rue Gaston Crémieux, CP5706, F-91057 Evry Cedex, France, Tel.: 33 +1 60 87 25 37;
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Fax: 33 +1 60 87 25 14; E-mail: fischer@genoscope.cns.fr
To whom correspondence should be addressed: Cécile Fischer, CEA-Institut de Génomique (IG),
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Supporting Information
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Figure S1. Complementation tests outline. The aim of this experiment is to exchange the selection
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marker cassette present in the ACIAD3540::KanR strain by a candidate gene. Primers fwd and rev
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were used to amplify the target gene. The primers P4 and P5 were extended by the complementary
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sequences of the integration gene amplification primers. Primers P3 and P6 for the amplification of the
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flanking regions were chosen to obtain regions of about 300±50 bp. The PCR products (gene and
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flanking regions R1 and R2) were then combined and amplified to generate a linear fragment
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containing the target gene flanked by two specific regions of A. baylyi ADP1. Upon recombination,
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PCR products generated by primers P7 and P8 were sequenced to check the correct locus integration
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and that no mutations had been introduced during the PCR process. External primers P7 and P8 were
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chosen to be 800±200 bp upstream and downstream of the cassette. The amplification, transformation
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and selection protocols were essentially as in de Berardinis et al.(2008).
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Figure S2. Growth of A. baylyi ADP1 wild-type and complemented strains. Microwell plates
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containing triplicate cultures grown aerobically in MAN-U (1 mM) medium at 30°C were monitored
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every 15 minutes for 24 hours on the Bioscreen (Thermo Scientific Labsystems). ACIAD3540::KanR
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(referred as to KO3540) is impaired in growth in contrast to the wild-type (WT ACIAD3540) and
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complemented strains (XCC0279, ACIAD3540, Mvan_5278, and tpucL).
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Figure S3. Steady state kinetics of urate oxidation in the presence of NADH or NADPH by XCC0279
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and the HpxO proteins ACIAD3540 and Mvan_5278. Plots A1-A3: Rates of urate oxidation versus
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urate concentrations. In plot A1, NADPH is used as the cosubstrate and in plots A2 and A3, NADH is
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used as the cosubstrate. Plots B1-B3: rates of urate oxidation versus NADH concentrations. Plots C1-
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C3: rates of urate oxidation versus NADPH concentrations. v is expressed in mole of urate oxidized
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per minute and per mole of enzyme. In main plots, curves were drawn using the Sigma-Plot program.
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The Michaelis-Menten equation v = (Vmax S)/(Km + S) applies for plots A1, A2, A3, B3, C1 and C3 and
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the Hill equation v = (Vmax Sn)/(S50n + Sn) for plots B1, B2, and C2. Insets show the Eadie-Hofstee
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representation (v versus v/S) of the kinetics. XCC0279 exhibits a Michaelian behaviour toward urate,
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NADH and NADPH while ACIAD3540 and Mvan_5278 exhibit an Michaelian behaviour only toward
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urate and a cooperative behaviour toward the reduced nicotinamide nucleotide (as unambiguously
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shown by the non-linearity of the Eadie-Hofstee plot (v versus v/S)
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Table S1. Oligonucleotide primers used in this study
Primers
Sequences
Genomic complementation
P3: fwd
P4_XCC0279: rev
P4_ACIAD3540: rev
P4_Mvan_5278: rev
P4_tpucL: rev
P5_XCC0279: fwd
P5_ ACIAD 3540 fwd
P5_Mvan_5278: fwd
P5_tpucL: fwd
P6: rev
P7: fwd
P8: rev
5’-GTCTTCGACAAAAATAGGTGC-3’
5’-CAGCGCGTGTTCGAGTTGAGCCAGGCTCATGTCTTACTCCTTATTTTTACTAC-3’
5’-CATACCTGCACCAATAATGACCACATTCATGTCTTACTCCTTATTTTTACTAC-3’
5’-CATTCCCGCACCGACGATCACGACCTTCATGTCTTACTCCTTATTTTTACTAC-3’
5’-TCCTTTGCCATAAGACATGGTTCTTTTCATGTCTTACTCCTTATTTTTACTAC-3’
5’-TGGCCACAGCCCACGCAGGCGGTGGCATAATATTGTAGGCAACCCGCTCG-3’
5’-AGTAATATTGTAGGCAACCCGCTCGATTAATATTGTAGGCAACCCGCTCG-3’
5’-GACTCCCGTCACCGCCGCTACCGAGGGCTAATATTGTAGGCAACCCGCTCG-3’
5’-GCTGAAAAATGTCGGAGCCTGAAAGCCTAATATTGTAGGCAACCCGCTCG-3’
5’-TATCGTGCCAATAACGAAAG-3’
5’-ACCTGTGAAAAAAGTGGTATCG-3’
5’-CCTATTTTGAGTTATCACCACG-3’
XCC0279: fwd
XCC0279: rev
ACIAD3540 fwd
ACIAD3540: rev
Mvan_5278: fwd
Mvan_5278: rev
tpucL: fwd
tpucL: rev
5’-ATGAGCCTGGCTCAACTCGAA-3’
5’-TTATGCCACCGCCTGCGT-3’
5’-ATGAATGTGGTCATTATTGGTGCA-3’
5’-TTAATCGAGCGGGTTGCCTAC-3’
5’-ATGAAGGTCGTGATCGTCGGT-3’
5’-TTAGCCCTCGGTAGCGGC-3’
5’-ATGAAAAGAACCATGTCTTATGGC-3’
5’-TTAGGCTTTCAGGCTCCGACA-3’
Recombinant proteins production.
XCC0279: fwd
XCC0279: rev
ACIAD3540: fwd
ACIAD3540: rev
Mvan_5278: fwd
Mvan_5278: rev
check 1: fwd
check 2: rev
5’-AAAGAAGGAGATAGGATCATGCATCATCACCATCACCATAGCCTGGCTCAACTCGAACAC-3’
5’-GTGTAATGGATAGTGATCTCATGCCACCGCCTGCGTGGGCTG-3’
5’-AAAGAAGGAGATAGGATCATGCATCATCACCATCACCAT-3’
5’- GTGTAATGGATAGTGATCTCA-3’
5’-AAAGAAGGAGATAGGATCATGCATCATCACCATCACCATAAGGTCGTGATCGTCGGTGCG-3’
5’-GTGTAATGGATAGTGATCTCAGCCCTCGGTAGCGGCGGT-3’
5’-ATCGAGATCTCGATCCCGCG-3’
5’-GCAGCAGCCAACTCAGCTTCC-3’
Substitution in primers in comparison to the original sequence are in bold: ATG replaced any occurring GTG codon
and stop codon was uniformely set to TAA. tpucL encodes the C-terminal domain (aa [171-end]) of B. subtilis PucL.
Cloning and expression of the recombinant proteins were done in the E. coli Top10 and BL21 (DE3) pLysS strains,
respectively (Invitrogen) using a modified Novagen pET22b(+) vector allowing directional cloning using the
ligation independent cloning method (de Jong et al., 2006).
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Table S2. Steady state kinetics of urate oxidation by the HpxO proteins ACIAD3540 and Mvan_5278
Enzyme
Substrate
Michaelian parameters
Non-Michaelian parameterse
kcat/Km
Km (µM)
kcat (s-1)
S50 (µM)
Vmax (s-1)
n
n'f
(s-1. M-1)
ACIAD3540 NADHa
118 ± 13 25.7 ± 2.0 2.0 ± 0.3 1.5 ± 0.1
NADPHa 511 ± 74 0.71 ± 0.05
1.4 103
Urateb
76 ± 7
23.6 ± 0.80
3.1 105
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Mvan_5278
NADH
265 ± 26 47.8 ± 3.6 2.4 ± 0.4 2.0 ± 0.1
NADPHc
271 ± 35 2.3 ± 0.2 2.3 ± 0.5 1.5 ± 0.1
Urated
413 ± 48 56.0 ± 2.50
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The urate concentration was 250 µM.
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The NADH concentration was 280 µM.
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The urate concentration was 1.5 mM.
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The NADH concentration was 600 µM.
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S50 is the substrate concentration showing half-maximal velocity, n is the Hill coefficient, and Vmax is the maximal
velocity. Each value represents the parameter determined by the non-linear least squares method.
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n’ is the Hill coefficient determined by the linear least squares method, determined from plots of log[v/(Vmax - v]
versus log S (Hill plot) using linear regression
The recombinant proteins were produced and purified according to the procedure used for XCC0279, as were the
enzymatic assays (see main text). Mvan_5278 is a NADH-preferring enzyme, as the Vmax obtained with NADH is
more than 20 times higher in the presence of NADH with a S50 value similar for both cosubstrates.
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Supplementary Text. Kinetic characterization of the HpxO proteins ACIAD3540 and Mvan_5278.
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In this section are provided and discussed kinetic parameters of two HpxO proteins giving further
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insights into the HpxO family
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Kinetic characterization of ACIAD3540: Kinetics of urate oxidation conducted with a fixed urate
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concentration and increasing NADPH concentrations, and those conducted with a fixed NADH
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concentration and a varying concentration of urate were hyperbolic (see also Figure S3, plots A2 and
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C2 respectively; the Michaelian behaviour of ACIAD3540 is further illustrated by the linearity of the
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Eadie-Hofstee representation (v versus v/S) in the insets). On the opposite, kinetics of urate oxidation
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conducted with a fixed urate concentration and increasing NADH concentrations were sigmoidal (see
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also Figure S3, plot B2; the cooperative behaviour of ACIAD3540 for NADH is unambiguously
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confirmed in the inset by the non-linearity of the Eadie-Hofstee plot (v versus v/S)). In this case values
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of v at various substrate concentrations were fit using non-linear regression, according to the general
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Hill equation (Equation 1):
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v = (Vmax Sn)/(S50n + Sn)
(1)
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where S50 is the substrate concentration showing a half maximal velocity, n is the Hill coefficient, and
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Vmax is the maximal velocity. A careful inspection of the kinetics presented in the panel C2 of Figure
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S3 may permit to suspect a sigmoidal cooperative effect for NADPH. However, fitting of these data
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according to the Hill equation gave values for n of 1.2 ± 0.1, and 1.1 ± 0.1 (using non linear and linear
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regressions, respectively), which rather suggest that the enzyme has indeed a Michaelian behaviour for
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this nucleotide.The binding of one molecule of NADH to one subunit of the ACIAD3540 dimer may
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cause a conformational change that increases the binding affinity of the cosubstrate for the second
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subunit, leading to positive homotropic cooperativity, as described for the well-studied prototype
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protein of the single-component monooxygenase family, the FAD-dependent monooxygenase
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p-hydroxybenzoate hydroxylase (PHBH) (reviewed in (Entsch and van Berkel, 1995)).
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Kinetic characterization of Mvan_5278: While Michaelis-Menten kinetics were observed when the
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concentration of urate varied in the presence of saturating NADH levels (see also Figure S3, plot A3),
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cooperativity was observed with respect to NADH and NADPH (see also Figure S3, plots B3, and C3
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respectively. This was unexpected, however, since Mvan_5278 appears experimentally to be a
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monomeric protein. A number of monomeric enzymes have been described as cooperative (Cornish-
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Bowden and Cardenas, 1987). Different kinetic models may account for this behaviour, as in the
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mnemonic (Ricard et al., 1974) and the ligand-induced slow transition (Ainslie et al., 1972) models.
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References
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Ainslie, G.R., Jr., Shill, J.P., and Neet, K.E. (1972) Transients and cooperativity. A slow transition
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model for relating transients and cooperative kinetics of enzymes. J Biol Chem 247: 7088-7096.
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Cornish-Bowden, A., and Cardenas, M.L. (1987) Co-operativity in monomeric enzymes. J Theor Biol
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124: 1-23.
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Entsch, B., and van Berkel, W.J. (1995) Structure and mechanism of para-hydroxybenzoate
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hydroxylase. FASEB J 9: 476-483.
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Ricard, J., Meunier, J.C., and Buc, J. (1974) Regulatory behavior of monomeric enzymes. 1. The
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mnemonical enzyme concept. Eur J Biochem 49: 195-208.
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