Abstract
The high activity of urease, a Ni(ii) enzyme, has several adverse effects on human health and agriculture, and its modulation needs the use of inhibitors. 1,4-Benzoquinone (BQ) irreversibly inactivates Sporosarcina pasteurii urease (SPU), with first order kinetics for both the inhibitor and the enzyme. This reaction is stoichiometrically quenched in the presence of sulphite. The 2.07 Å crystal structure of SPU bound to BQ shows the presence of a 1,4-hydroquinone moiety covalently bound to the thiol group of αCys322, a key residue found on the mobile flap regulating the substrate access to the active site. The 1.75 Å crystal structure obtained when sulphite is added to a solution of SPU previously incubated with BQ shows the presence of a 2,5-dihydroxy-benzenesulphonate moiety bound to the αCys322 thiol group. These data reveal how the active site cysteine reacts with a prototypical BQ moiety, found in a large number of natural substances potentially suitable to control the urease activity.
Highlights
The high activity of urease, a Ni(II) enzyme, has several adverse effects on human health and agriculture, and its modulation needs the use of inhibitors. 1,4-Benzoquinone (BQ) irreversibly inactivates Sporosarcina pasteurii urease (SPU), with first order kinetics for both the inhibitor and the enzyme
The 2.07 Å crystal structure of SPU bound to BQ shows the presence of a 1,4-hydroquinone moiety covalently bound to the thiol group of αCys[322], a key residue found on the mobile flap regulating the substrate access to the active site
Covalent modifications of this residue by the formation of a disulphide bond in the presence of 2-mercaptoethanol, demonstrated by crystallography,[10] lead to enzyme inhibition.[11]. These observations support the hypothesis that the conserved cysteine in the flap is the target for quinone-based inhibitors, as shown in Scheme 1, by the formation of 2.7d Here, we describe a kinetic characterization of the inhibition of SPU by 1,4-benzoquinone, and report, for the first time, the high-resolution crystal structure of the urease–BQ covalent complex, shedding light on the longawaited molecular details of this interaction
Summary
1,4-benzoquinone moiety (BQ, 1, Scheme 1).[7] Quinones are known to react with thiols, leading to the formation of the corresponding thiol-substituted benzene-1,4-diol.[8] The sequence of the urease mobile flap contains the highly conserved residue αCys[322] (residue numbering as in SPU), which is crucial for the activity of urease,[9] and whose position with respect to the active site is modulated by the flap movement (Fig. 1B) Covalent modifications of this residue by the formation of a disulphide bond in the presence of 2-mercaptoethanol, demonstrated by crystallography,[10] lead to enzyme inhibition.[11] These observations support the hypothesis that the conserved cysteine in the flap is the target for quinone-based inhibitors, as shown in Scheme 1, by the formation of 2.7d Here, we describe a kinetic characterization of the inhibition of SPU by 1,4-benzoquinone, and report, for the first time, the high-resolution crystal structure of the urease–BQ covalent complex, shedding light on the longawaited molecular details of this interaction. The resulting 2.07 Å crystal structure of SPU inhibited by BQ (see Table 1 and the ESI† for full details of crystallization, X-ray data collection, elaboration and analysis) confirmed the wellestablished architecture of the enzyme, as well as the rigidity of the protein scaffold, with a root mean square deviation (RMSD) for the Cα residue atoms of only 0.12 Å with respect to the structure of native urease (PDB code 4CEU)
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