Publications

• Azole drugs trap cytochrome P450 EryK in alternative conformational states.

  Montemiglio L.C., Gianni S., Vallone B., Savino C.

  EryK is a bacterial cytochrome P450 that catalyzes the last hydroxylation occurring during the biosynthetic pathway of erythromycin A in Streptomyces erythraeus. We report the crystal structures of EryK in complex with two widely used azole inhibitors: ketoconazole and clotrimazole. Both of these ... >> More

  EryK is a bacterial cytochrome P450 that catalyzes the last hydroxylation occurring during the biosynthetic pathway of erythromycin A in Streptomyces erythraeus. We report the crystal structures of EryK in complex with two widely used azole inhibitors: ketoconazole and clotrimazole. Both of these ligands use their imidazole moiety to coordinate the heme iron of P450s. Nevertheless, because of the different chemical and structural properties of their N1-substituent group, ketoconazole and clotrimazole trap EryK, respectively, in a closed and in an open conformation that resemble the two structures previously described for the ligand-free EryK. Indeed, ligands induce a distortion of the internal helix I that affects the accessibility of the binding pocket by regulating the kink of the external helix G via a network of interactions that involves helix F. The data presented thus constitute an example of how a cytochrome P450 may be selectively trapped in different conformational states by inhibitors. << Less

  Biochemistry 49:9199-9206(2010) [PubMed] [EuropePMC]

• Overproduction and characterization of the erythromycin C-12 hydroxylase, EryK.

  Lambalot R.H., Cane D.E., Aparicio J.J., Katz L.

  Hydroxylation of C-12 is one of the final steps in the biosynthesis of erythromycin A (ErA). A point of uncertainty in the erythromycin pathway has been whether the C-12 hydroxylase operates on each of two possible substrates, erythromycin B (ErB) and erythromycin D (ErD). Stassi et al. have clone ... >> More

  Hydroxylation of C-12 is one of the final steps in the biosynthesis of erythromycin A (ErA). A point of uncertainty in the erythromycin pathway has been whether the C-12 hydroxylase operates on each of two possible substrates, erythromycin B (ErB) and erythromycin D (ErD). Stassi et al. have cloned the gene, designated eryK, which encodes the P-450 monooxygenase responsible for erythromycin C-12 hydroxylation in Saccharopolyspora erythraea [Stassi, D., Donadio, S., Staver, M. J., & Katz, L. (1993) J. Bacteriol. 175, 182-189]. We report the overproduction of EryK in Escherichia coli as insoluble inclusion bodies; the solubilization, refolding, and reconstitution of active holo-EryK; and kinetic confirmation of a 1200-1900-fold preference of the enzyme for ErD over the alternative C-12 hydroxylase substrate ErB. Our results indicate that ErB is a shunt metabolite in the erythromycin biosynthetic pathway. << Less

  Biochemistry 34:1858-1866(1995) [PubMed] [EuropePMC]

• Investigating the structural plasticity of a cytochrome P450: three-dimensional structures of P450 EryK and binding to its physiological substrate.

  Savino C., Montemiglio L.C., Sciara G., Miele A.E., Kendrew S.G., Jemth P., Gianni S., Vallone B.

  Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of su ... >> More

  Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism. << Less

  J. Biol. Chem. 284:29170-29179(2009) [PubMed] [EuropePMC]