Fig 1.
Purification and pyrophosphatase activity of PHP domain.
A) SDS-PAGE of fractions collected during the purification of the E. coli PHP domain. M: molecular mass markers. Lane 1: soluble proteins isolated after refolding of the inclusion bodies containing PHP; lanes 2 and 3: purified PHP domain after ion exchange and gel filtration chromatography, respectively. B) Hydrolysis of pyrophospate by PHP domain. The release of orthophosphate as a function of time is reported for a complete assay mixture (green dots, 176 nM PHP and 1 mM pyrophosphate), and for assay solutions lacking pyrophosphate or enzyme (blue and red dots, respecitvely). C) Pyrophosphatase activity of 0.45 nM E. coli inorganic pyrophosphatase in the absence (green dots) or in the presence of 40, 100, 200, or 800 μM NaF (blue, red, cyano, and dark red dots, respectively). D) Pyrophosphatase activity of 30 nM full-length α subunit in the absence (green dots) or in the presence of 50, 200, or 800 μM NaF (blue, red, and cyano dots, respectively).
Fig 2.
Reaction steps leading to the production of uric acid at the expense of phosphate and inosine.
Fig 3.
Structures of E. coli PHP and type-II inorganic PPases.
A) Tertiary structure of E. coli DNA Polymerase III α subunit (PDB 2HNH). The PHP and the Polymerase domains are represented in green and blue, respectively. B,C) Detail of the entire PHP domain (B) and of the PHP β-sheet (C). The antiparallel β-strand is represented in C with green colour. D,E) Active sites of the type-II inorganic PPase from Bacillus subtilis (D, PDB 1WPM) and Streptococcus gordonii (E, PDB 1K20). The proposed active site of E. coli PHP is shown in panel F. The following amino acids are shown as sticks: H9, D13, D15, D75, H98, and D149 (D, Bacillus subtilis); H9, D13, D15, D77, H99, and D151 (E, Streptococcus gordonii); H12, D19, D43, D69, H83, and D201 (F, Escherichia coli PHP).
Fig 4.
Pyrophosphatase and DNA Polymerase activity of wt and site-specific variants of α subunit.
A) Pyrophosphatase activity of 17 nM wt (green dots), 17 nM H12A (blue dots), or 13 nM D19A (red dots) α subunit. B,C) DNA Polymerase activity as determined in the presence (B) or in the absence of E. coli inorganic pyrophosphatase (C). Other conditions as in A. D) Pyrophosphatase activity of 4.5 nM τ3α3ε3θ3 (green dots), or τ3α(D201A)3ε3θ3 complex (red dots). E,F) DNA Polymerase activity as determined in the presence (E) or in the absence of E. coli inorganic pyrophosphatase (F). The concentration of assayed enzymes were 5 and 3.2 nM for the wt and the τ3α(D201A)3ε3θ3 complex, respectively.
Fig 5.
Observed rate constants of wt and site-specific variants of α subunit.
Observed rate constants (v/[Et]) for the hydrolysis of pyrophosphate (PPi), and for DNA replication in the absence (Pol–PPase) or in the presence (Pol + PPase) of inorganic PPase. For the τ3α3ε3θ3 and τ3α(D201A)3ε3θ3 complex, rate constants are reported per monomer. The error bars represent, except for αD19A, the standard deviations of 3 independent kinetic assays, performed with different enzyme preparations. The purification yield of αD19A was not sufficient to perform triplicate assays. In this case, the error bars represent the standard deviations of the best linear fits to the different observed kinetics.
Fig 6.
Phenotype of E. coli overexpressing or not the wt, D201A, or the H12A α subunit.
Bright field (A,C,E,G,I,M) and fluorescence (B,D,F,H,L,N) micrographs of cells not-induced (A,B,E,F,I,L) or subjected (C,D,G,H,M,N) to overexpression of wt (A-D), D201A (E-H), or H12A (I-N) α subunit.
Fig 7.
Growth kinetics of E. coli overexpressing or not the wt, D201A, or the H12A α subunit.
A,B) Growth kinetics, determined spectroscopically, of E. coli populations overexpressing (filled symbols) or not (empty symbols) the wt (circles), D201A (squares, A), or the H12A (squares, B) α subunit. C,D) Growth kinetics, determined by colony counting, of E. coli populations overexpressing (filled symbols) or not (empty symbols) the wt (circles), D201A (squares, C), or the H12A (squares, D) α subunit.