Catalytic properties of catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 immobilized on nanosponges (original) (raw)
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Journal of protein chemistry, 2000
Two catechol 1,2-dioxygenase (C1,2O) isozymes (IsoA and IsoB) have been purified to homogeneity from a strain of Acinetobacter radioresistens grown on benzoate as the sole carbon and energy source. IsoA and IsoB are both homodimers composed of a single type of subunit with molecular mass of 38,600 and 37,700, Da respectively. In conditions of low ionic strength, IsoA can aggregate as a trimer, in contrast to IsoB, which maintains the dimeric structure, as also supported by the kinetic parameters (Hill numbers). IsoA is identical to the enzyme previously purified from the same bacterium grown on phenol, whereas the IsoB is selectively expressed using benzoate as carbon source. This is the first evidence of the presence of differently expressed C1,2O isozymes in A. radioresistens or more generally of multiple C1,2O isozymes in benzoate-grown Acinetobacter cells. Purified IsoA and IsoB contain approximately 1 iron(III) ion per subunit and both show electronic absorbance and EPR feature...
FEBS Letters, 1997
A catechol 1,2-dioxygenase (Cl,20) has been purified to homogeneity from Acinetobacter radioresistens grown on phenol as the sole carbon and energy source. The Cl,20 appears to be a homodimer, with a molecular mass of 78000 Da. At relatively high ionic strengths (0.5 M Na 2 S0 4 ) subunit dissociation occurs and the monomeric unit (38 700 Da) is shown to be active. This phenomenon has never been observed before in dioxygenases. The purified Cl,20 contains 0.96 iron(III) ions per unit and spectroscopic measurements suggest the presence of one high-spin iron(III) ion in an environment characteristic of intradiol cleaving enzymes. The NH 2 -terminal amino acid sequence has been determined and compared to the primary structures of intradiol rings cleaving dioxygenases from other Acinetobacter strains revealing 45% homology with the benzoate-grown A. calcoaceticus ADP-1 and an identity of only one of the 20 amino acids sequenced for the phenol-grown A. calcoaceticus NCIB 8250.
Research in Microbiology, 2002
Two novel catechol 1,2-dioxygenase (C 1,2-O) genes have been isolated from an Acinetobacter radioresistens strain that grows on phenol or benzoate as sole carbon and energy source. Designated as catA A and catA B , they encode proteins composed of 314 and 306 amino acids, whose deduced sequences indicate that they have approximately 53% identity, whereas their NH 2 -terminal and COOH-terminal regions have no sequences in common. This may explain their different thermal and pH stability. Polyclonal antibodies raised against an amino-terminal CatA A peptide or the whole CatA B protein were used to establish their inducible and differential expression patterns upon bacterial growth in phenol or benzoate. The CatA A protein (IsoA) was induced by both phenol and benzoate though with different kinetics, whereas the catA B product (IsoB) was constitutively produced at low levels that increased only during growth in the presence of benzoate. 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2011
Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Journal of Structural Biology, 2010
The first crystallographic structures of a catechol 1,2-dioxygenase from a Gram-positive bacterium Rhodococcus opacus 1CP (Rho 1,2-CTD), a Fe(III) ion containing enzyme specialized in the aerobic biodegradation of catechols, and its adducts with catechol, 3-methylcatechol, 4-methylcatechol, pyrogallol (benzene-1,2,3-triol), 3-chlorocatechol, 4-chlorocatechol, 3,5-dichlorocatechol, 4,5-dichlorocatechol and protocatechuate (3,4-dihydroxybenzoate) have been determined and analyzed.
Archives of Biochemistry and Biophysics, 2004
The reversible active site metal ion removal process for two catechol 1,2-dioxygenase isoenzymes (IsoA and IsoB) isolated from Acinetobacter radioresistens S13 has been monitored using circular dichroism and fluorescence spectroscopic techniques. IsoA and IsoB are homodimers, containing one iron(III) ion per subunit. Their amino acid sequence identity is 48.4%. Previous experiments suggested that structural diversities could be responsible for the differential thermal and pH stabilities of the two isoenzymes and of their distinct demetallation kinetics. The far-UV CD spectra of IsoA and IsoB catechol 1,2-dioxygenases from A. radioresistens S13 provide information on their secondary structures. IsoB appears to have a content of a-helices higher than IsoA. Upon metal ion removal, both proteins reversibly lose part of their secondary structure following distinct pathways. CD spectra simulations allowed us to estimate the content of a-helices, b-sheets, and turns for each isoenzyme and to monitor the secondary structure rearrangements. The metal ion withdrawal has large influence on the secondary structure: in particular a significant reduction of a-helices content is observed for both isoenzymes. Intrinsic fluorescence emission spectra clearly support such results, adding information on the local environment changes of the tryptophan residues. The positioning of Trp250 in IsoB has been shown to be of particular interest for monitoring the local structure changes occurring upon metal ion removal. For the first time these studies allow to underline the role of active site iron ions on dioxygenases folding and stability, further evidencing the differences in structural assembling between the two isoenzymes from A. radioresistens S13.
Enzyme and Microbial Technology, 2000
Thermophilic catechol 2,3-dioxygenase (EC 1.13.11.2) from Bacillus stearothermophilus has been immobilized on highly activated glyoxyl agarose beads. The enzyme could be fully immobilized at 4°C and pH 10.05 with a high retention of activity (around 80%). Enzyme immobilized under these conditions showed little increase in thermostability compared with the soluble enzyme, but further incubation of immobilized enzyme at 25°C and pH 10.05 for 3 h before borohydride reduction resulted in conjugates exhibiting a 100-fold increase in stability (c.f. the free enzyme). The stability of catechol 2,3-dioxygenase immobilized under these conditions was essentially independent of protein concentration whereas free enzyme was rapidly inactivated at low protein concentrations. An apparent stabilization factor of over 700-fold was recorded in the comparison of free and immobilized catechol 2,3-dioxygenases at protein concentrations of 10 g/ml. Immobilization increased the 'optimum temperature' for activity by 20°C, retained activity at substrate concentrations where the soluble enzyme was fully inactivated and enhanced the resistance to inactivation during catalysis. These results suggest that the immobilization of the enzyme under controlled conditions with the generation of multiple covalent links between the enzyme and matrix both stabilized the quaternary structure of the protein and increased the rigidity of the subunit structures.
Intersubunit interaction and catalytic activity of catechol 2,3-dioxygenases
Biochemical Journal, 2003
Catechol 2,3-dioxygenases (C23Os; EC 1.3.11.2) form a large protein family that is divided into several subgroups. Amino acid sequences of C23Os belonging to subgroup I.2.A and those belonging to I.2.B are found to be approx. 50% identical. When the central parts of the C23O sequences belonging to I.2.B were fused with the N-terminal and C-terminal sequences of I.2.A C23O, the hybrid enzymes were not active. To understand why these hybrid C23Os were inactive, hybrids between XylEP (C23O found in a Pseudomonas strain; subgroup I.2.A) and XylES (C23O found in a Sphingomonas strain; subgroup I.2.B) were constructed. HB3-C23O consisted mostly of the XylES sequence, except that its C-terminal end was derived from XylEP. While HB3-C23O was not active, HB4-C23O, carrying shorter C-terminal XylEP sequences than HB3-C23O, was active. This observation indicated that certain amino acid residues at the C-terminus were crucial for C23O activity in the hybrid forms of enzymes between XylEP and Xy...