In vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of aromatic degradative pathways (original) (raw)
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Journal of bacteriology, 1994
Catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida consists of four identical subunits, each containing one ferrous ion. The enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatechol. Two mutants of catechol 2,3-dioxygenases (4ECR1 and 4ECR6) able to oxidize 4-ethylcatechol, one mutant (3MCS) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and 4-methylcatechol, and one phenotypic revertant of 3MCS (3MCR) which had regained the ability to oxidize 3-methylcatechol were characterized by determining their Km and partition ratio (the ratio of productive catalysis to suicide catalysis). The amino acid substitutions in the four mutant enzymes were also identified by sequencing their structural genes. Wild-type catechol 2,3-dioxygenase was inactivated during the catalysis of 4-ethylcatechol and thus had a low partition ratio for this subs...
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...
Applied and Environmental Microbiology, 2004
Catechol 2,3-dioxygenase (C23O; EC 1.3.11.2), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols. C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III). In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated. To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence ( xylE or nahH ) to construct a library of chimeric C23O genes. These chimeric C23O genes were then introduced into Pseudomonas putida possessing some of the toluene catabolic genes ( xylXYZLGFJQKJI ). When a C23O gene (e.g., xylE ) is introduced into this strain, the transformants cannot generally grow on p -toluate because 4-methylcatechol, a metabolite of p -toluate, is a substrate as well as a suici...
Activity of a carboxyl-terminal truncated form of catechol 2,3-dioxygenase from Planococcus sp. S5
TheScientificWorldJournal, 2014
Catechol 2,3-dioxygenases (C23Os, E.C.1.13.12.2) are two domain enzymes that catalyze degradation of monoaromatic hydrocarbons. The catalytically active C-domain of all known C23Os comprises ferrous ion ligands as well as residues forming active site pocket. The aim of this work was to examine and discuss the effect of nonsense mutation at position 289 on the activity of catechol 2,3-dioxygenase from Planococcus strain. Although the mutant C23O showed the same optimal temperature for activity as the wild-type protein (35 °C), it exhibited activity slightly more tolerant to alkaline pH. Mutant enzyme exhibited also higher affinity to catechol as a substrate. Its K(m) (66.17 µM) was approximately 30% lower than that of wild-type enzyme. Interestingly, removal of the C-terminal residues resulted in 1.5- to 1.8-fold (P < 0.05) increase in the activity of C23OB61 against 4-methylcatechol and 4-chlorocatechol, respectively, while towards catechol the activity of the protein dropped to ...
Gene, 1994
Pseudomonas sp. strain HV3 degrades aromatics and chloroaromatics. It harbours a mega-plasmid, designated pSKY4, from which the gene cmpE, encoding a catechol 2,3-dioxygenase (C230) catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde, was cloned and sequenced. The deduced amino acid (aa) sequence shows the highest homology, 52%, to the deduced aa sequences of xylE1 and dmpB. The deduced 307-aa sequence of cmpE contains the extradiol ring-cleavage signature in the same position as other 307-aa C230-encoding genes.
Catechol 1,2-dioxygenase from the new aromatic compounds – Degrading Pseudomonas putida strain N6
International Biodeterioration & Biodegradation, 2011
This study aimed to characterization of catechol 1,2-dioxygenase from a Gram-negative bacterium, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. Strain designated as N6, was isolated from the activated sludge samples of a sewage treatment plant at Bentwood Furniture Factory Jasienica, Poland. Morphology, physio-biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicate that strain belongs to Pseudomonas putida. When cells of strain N6 grown on protocatechuate or 4-hydroxybenzoic acid mainly protocatechuate 3,4-dioxygenase was induced. The activity of catechol 1,2-dioxygenase was rather small. The cells grown on benzoic acid, catechol or phenol showed high activity of only catechol 1,2-dioxygenase. This enzyme was optimally active at 35 C and pH 7.4. Kinetic studies showed that the value of K m and V max was 85.19 mM and 14.54 mM min À1 respectively. Nucleotide sequence of gene encoding catechol 1,2-dioxygenase in strain N6 has 100% identity with catA genes from two P. putida strains. The deduced 301-residue sequence of enzyme corresponds to a protein of molecular mass 33.1 kDa. The deduced molecular structure of the catechol 1,2dioxygenase from P. putida N6 was very similar and characteristic for the other intradiol dioxygenases.