Abstract
The different behaviour of two isozymes (IsoA and IsoB) of catechol 1,2-dioxygenase (C1,2O) from Acinetobacter radioresistenss13 on a hydrophobic interaction, Phenyl-Sepharose chromatographic column, prompted us to investigate the role of superficial hydrophobicity on structural-functional aspects for such class of enzymes. The interaction of 8-anilino-1-naphtalenesulphonate (ANS), a fluorescent probe known to bind to hydrophobic sites in proteins, revealed that the two isoenzymes have a markedly different hydrophobicity degree although a similar number of hydrophobic superficial sites were estimated (2.65 for IsoA and 2.18 for IsoB). ANS is easily displaced by adding the substrates catechol or 3-methylcatechol to the adduct, suggesting that the binding sites are in the near surroundings of the catalytic clefts. The analysis of the hydropathy profiles and the possible superficial cavities allowed to recognize the most feasible region for ANS binding.
The lower hydrophobicity detected in the near surroundings of the catalytic pocket of IsoB supports its peculiarity to lose the catalytic metal ions more easily than IsoA. As previously suggested for other metalloenzymes, the presence of more hydrophilic and/or smaller residues near to the active site of IsoB is expected to increase the metal ligands mobility thus increasing the metal ion dissociation rate constants, estimated to be 0.078 h−1 and 0.670 h−1 for IsoA and IsoB respectively.
Abbreviations: C1,2O – catechol dioxygenase; ANS – 8-anilino-1-naphthalenesulphonate; PHO – phenol hydroxylase oxygenase
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aoki K, Konohana T, Shinke R, Nishira H. 1984 Two catechol 1,2-dioxygenase from an aniline-assimilating bacterium, Frateuria species ANA-18. Agric Biol Chem 48, 2097–2104.
Ashmore PG. 1979 Principles of reaction kinetics Revised 2nd edition. London: The Chemical Society
Black SD, Mould DR. 1991 Development of hydrophobicity para-meters to analyze proteins which bear post-or cotranslational modifications. Anal Biochem 193, 72–82.
Brand L. Gohlke JR. 1972 Fluorescence probes for structure. Annu Rev Biochem 41, 843–868.
Briganti F, Pessione E, Giunta C, Scozzafava A. 1997 Purifica-tion, biochemical properties and substrate specificity of a cat-echol 1,2-dioxygenase from a phenol degrading Acinetobacter radioresistens. FEBS Lett 416, 61–64.
Briganti F, Mangani S, Pedocchi L, Scozzafava A, Golovleva LA, Jadan AP, Solyanikova IP 1998 XAS characterization of the active sites of novel intradiol ring-cleaving dioxygenases: hy-droxyquinol and chlorocatechol dioxygenases. FEBS Lett 433, 58–62.
Briganti F, Pessione E, Giunta C, Mazzoli R, Scozzafava A. 2000 Purification and catalytic properties of two catechol 1,2-dioxygenase isozymes from benzoate-grown cells of Acinetobac-ter radioresistens. J Protein Chem 19, 709–716.
Caposio P, Pessione E, Giuffrida MG, Conti A, Landolfo S, Giunta C, Gribaudo G. 2002 Cloning and characterization of two cat-echol 1,2-dioxygenase genes from Acinetobacter radioresistenss13. Res Microbiol 153, 69–74.
Cardamone M, Puri NK. 1992 Spectrofluorimetric assessment of the surface hydrophobicity of proteins. Biochem J 282, 589–593.
Divari S, Valetti F, Caposio P, Pessione E, Cavaletto M, Griva E, Gribaudo G, Gilardi G, Giunta C. 2003 The oxygenase com-ponent of phenol hydroxylase from Acinetobacter radioresistens S13. Eur J Biochem 270, 2244–2253.
Giuffrida MG, Pessione E, Mazzoli R, Dellavalle G, Barello C, Conti A, Giunta C. 2001 Media containing aromatic compounds induce peculiar proteins in Acinetobacter radioresistens, as re-vealed by proteome analysis. Electrophoresis 22, 1705–1711.
Hunt JA, Ahmed M, Fierke CA. 1999 Metal binding specificity in carbonic anhydrase is influenced by conserved hydrophobic core residues. Biochemistry 38, 9054–9062.
Kim SI, Kweon SM, Kim S, Ha, KS 1997 Peptide mapping and amino acid sequencing of two catechol 1,2-dioxygenases (CD I1 and CD I2) from Acinetobacter lwoffii K24. Mol. Cells 31, 635–640.
Murakami S, Wang CL, Naito A, Shinke R, Aoki, K 1998. Purification and characterization of four catechol 1,2-dioxygenase isozymes from the benzamide-assimilating bacterium Arthrobacter speciesBA–5–17. Microbiol Res 153, 163–171.
Nakai C, Horiike K, Kuramitsu S, Kagamiyama H, Nozaki M. 1990. Three isozymes of catechol 1,2-dioxygenase (pyrocatechase), alpha alpha, alpha beta, and beta beta, from Pseudomonas arvilla C-1. J Biol Chem 265, 660–665.
Ornston LN, Stanier RY 1966. The conversion of catechol and pro-tocatechuate to beta-etoadipate by Pseudomonas putida. J Biol Chem 241, 3776–3786.
Pessione E, Giuffrida MG, Mazzoli R, Caposio P, Landolfo S, Conti A, Giunta C, Gribaudo G. 2001. The catechol 1,2 dioxygenase system of Acinetobacter radioresistens: isoenzymes, inductors and gene localisation. Biol Chem 382, 1253–1261.
Pessione E, Giuffrida MG, Prunotto L, Barello C, Mazzoli R, Fortunato D, Conti A, Giunta C. 2003 Membrane proteome of Acinetobacter radioresistens S13 during aromatic exposure. Proteomics 3, 1070–1076.
Suzuki M, Hayakawa T, Shaw JP, Rekik M, Harayama S. 1991 Primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system. J Bacteriol 173, 1690–1695.
Thompson JD, Higgins DG, Gibson TJ. 1994 CLUSTAL W: im-proving the sensitivity of progressive multiple sequence align-ment through sequence weighting, position-specific gap penal-ties and weight matrix choice. Nucleic Acids Re 22, 4673–4680.
Tsodikov OV, Record MT Jr, Sergeev YV. 2002 Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature. J Comput Chem 23, 600–609.
Vetting MW, Ohlendorf DH 2000 The 1.8 A crystal structure of cat-echol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker. Structure Fold Des 8, 429–440.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Di Nardo, G., Pessione, E., Cavaletto, M. et al. Effects of surface hydrophobicity on the catalytic iron ion retention in the active site of two catechol 1,2-dioxygenase isoenzymes. Biometals 17, 699–706 (2004). https://doi.org/10.1007/s10534-004-1208-x
Issue Date:
DOI: https://doi.org/10.1007/s10534-004-1208-x