3,4-Dihydroxyphenylacetate 2,3-dioxygenase from <i>Klebsiella pneumoniae</i>, a Mg2+-containing dioxygenase involved in aromatic catabolism

Gibello, Alicia; Ferrer, Esther; Martín, Margarita; Garrido-Pertierra, Amando

doi:10.1042/bj3010145

Cited by 49 publications

(32 citation statements)

References 35 publications

(37 reference statements)

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“…Surprisingly, both dioxygenases did not present any similarity to other extradiol or intradiol dioxygenases sequenced so far. However, it is worth noting that the HPC 2,3-dioxygenase of K. pneumoniae M5a1, which has the peculiarity of requiring magnesium for activity (19), seems to be similar to HpaD and HpcB (Fig. 1).…”

Section: Resultsmentioning

confidence: 95%

Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster

1996

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We have determined and analyzed the nucleic acid sequence of a 14,855-bp region that contains the complete gene cluster encoding the 4-hydroxyphenylacetic acid (4-HPA) degradative pathway of Escherichia coli W (ATCC 11105). This catabolic pathway is composed by 11 genes, i.e., 8 enzyme-encoding genes distributed in two putative operons, hpaBC (4-HPA hydroxylase operon) and hpaGEDFHI (meta-cleavage operon); 2 regulatory genes, hpaR and hpaA; and the gene, hpaX, that encodes a protein related to the superfamily of transmembrane facilitators and appears to be cotranscribed with hpaA. Although comparisons with other aromatic catabolic pathways revealed interesting similarities, some of the genes did not present any similarity to their corresponding counterparts in other pathways, suggesting different evolutionary origins. The cluster is flanked by two genes homologous to the cstA (carbon starvation protein) and tsr (serine chemoreceptor) genes of E. coli K-12. A detailed genetic analysis of this region has provided a singular example of how E. coli becomes adapted to novel nutritional sources by the recruitment of a catabolic cassette. Furthermore, the presence of the pac gene in the proximity of the 4-HPA cluster suggests that the penicillin G acylase was a recent acquisition to improve the ability of E. coli W to metabolize a wider range of substrates, enhancing its catabolic versatility. Five repetitive extragenic palindromic sequences that might be involved in transcriptional regulation were found within the cluster. The complete 4-HPA cluster was cloned in plasmid and transposon cloning vectors that were used to engineer E. coli K-12 strains able to grow on 4-HPA. We report here also the in vitro design of new biodegradative capabilities through the construction of a transposable cassette containing the wide substrate range 4-HPA hydroxylase, in order to expand the ortho-cleavage pathway of Pseudomonas putida KT2442 and allow the new recombinant strain to use phenol as the only carbon source.Although most of our current knowledge about the general bacterial metabolic pathways has been derived from the analysis of Escherichia coli, very few data are available about the ability of this microorganism to grow on aromatic compounds other than amino acids. It has been shown that E. coli B, C, and W, but not K-12 strains, are able to degrade 4-hydroxyphenylacetic acid (4-HPA) and homoprotocatechuate (3,4-hydroxyphenylacetate) (HPC) via an inducible, chromosomally encoded meta-cleavage pathway (8, 10).The HPC degradative operon of E. coli C has been partially cloned (25,43), and some of its products have been characterized (16-18, 40-42, 44, 48). In addition, we have previously demonstrated that the first step in the 4-HPA degradation in E. coli W, i.e., the formation of HPC, is catalyzed by a twocomponent aromatic hydroxylase (38,39). This enzyme is encoded by two genes which appear to be part of the same operon (38). The homologous 4-HPA hydroxylase operon of E. coli C has been also cloned and partially sequenced (38...

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Section: Resultsmentioning

confidence: 95%

Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster

1996

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“…Both HpcB from E. coli C (268) and the equivalent HpaB protein from K. pneumoniae M5a1 (113) are highly unstable enzymes, and the addition of reagents such as glycerol, acetone, dithiothreitol, or iron salts, which are known to stabilize some ring cleavage dioxygenases, had no effect on these HPC dioxygenases. Unexpectedly, the HPC dioxygenase from K. pneumoniae appears to contain Mg 2ϩ instead of Fe 2ϩ in its tetrameric structure (113). Whether the HPC 2,3-dioxygenase from E. coli also requires Mg 2ϩ for its activity or requires Fe 2ϩ (like most extradiol dioxygenases) or Mn 2ϩ (like the HPC 2,3-dioxygenase from Arthrobacter globiformis [333]) remains to be determined.…”

Section: Aromatic Ring Cleavage Dioxygenasesmentioning

confidence: 99%

Biodegradation of Aromatic Compounds by Escherichia coli

Dı́az

Ferrández

Prieto

et al. 2001

Microbiol Mol Biol Rev

322

235

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Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications

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“…The three-dimensional structures of three Type I extradiol dioxygenases, two of which cleave bicyclic compounds (2, 3) and one of which cleaves monocyclic compounds (4), have been reported. Whereas a majority of the bacterial extradiol dioxygenases that have been characterized contain Fe(II) as a catalytic metal center, there are only three known bacterial 3,4-dihydroxyphenylacetate 2,3-dioxygenase that utilize metals other than Fe(II); the enzyme from Bacillus brevis (5) and Arthrobacter globiformis CM-2 (6) are manganese-dependent, whereas that from Klebsiella pneumoniae exhibits magnesium dependence (7).…”

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confidence: 99%

Characterization of a Novel Thermostable Mn(II)-dependent 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from a Polychlorinated Biphenyl- and Naphthalene-degrading Bacillus sp. JF8

Hatta

Mukerjee-Dhar

Damborský

et al. 2003

Journal of Biological Chemistry

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A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the metacleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp. JF8, and the gene was cloned. The native and recombinant BphC enzyme was purified to homogeneity. The enzyme has a molecular mass of 125 ؎ 10 kDa and was composed of four identical subunits (35 kDa). BphC_JF8 has a temperature optimum of 85°C and a pH optimum of 7.5. It exhibited a half-life of 30 min at 80°C and 81 min at 75°C, making it the most thermostable extradiol dioxygenase studied. Inductively coupled plasma mass spectrometry analysis confirmed the presence of 4.0-4.8 manganese atoms per enzyme molecule. The EPR spectrum of BphC_JF8 exhibited g ‫؍‬ 2.02 and g ‫؍‬ 4.06 signals having the 6-fold hyperfine splitting characteristic of Mn(II). The enzyme can oxidize a wide range of substrates, and the substrate preference was in the order 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-methylcatechol > 4-chlorocatechol. The enzyme is resistant to denaturation by various chelators and inhibitors (EDTA, 1,10-phenanthroline, H 2 O 2 , 3-chlorocatechol) and did not exhibit substrate inhibition even at 3 mM 2,3-dihydroxybiphenyl. A decrease in K m accompanied an increase in temperature, and the K m value of 0.095 M for 2,3-dihydroxybiphenyl (at 60°C) is among the lowest reported. The kinetic properties and thermal stability of the native and recombinant enzyme were identical. The primary structure of BphC_JF8 exhibits less than 25% sequence identity to other 2,3-dihydroxybiphenyl 1,2-dioxygenases. The metal ligands and active site residues of extradiol dioxygenases are conserved, although several amino acid residues found exclusively in enzymes that preferentially cleave bicyclic substrates are missing in BphC_JF8. A three-dimensional homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure, and stability of the enzyme.The catabolic versatility exhibited by microorganism plays an essential role in the carbon cycle, and this depends to a large extent on the use of oxygenases. In the degradation of aromatic compounds, oxygenases play a significant role both by hydroxylating the aromatic ring and by catalyzing the ring fission reaction. Nearly all bacterial pathways for the degradation of aromatic compounds transform initial substrates into intermediates that carry two or more hydroxyl groups on the aromatic ring, which are then substrates for the ring cleavage dioxygenases. Cleavage is generally catalyzed by metalloenzymes of one of the two functional classes: intradiol dioxygenases, which cleave ortho to the hydroxyl substituents, or extradiol dioxygenases, which cleave meta to the hydroxyl substituents.Harayama and Rekik (1) proposed that extradiol dioxygenases could be divided into two families, those exhibiting a preference for bicyclic substrates and those with a preference for monocyclic substrates. Since then, several ex...

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3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Klebsiella pneumoniae, a Mg2+-containing dioxygenase involved in aromatic catabolism

Cited by 49 publications

References 35 publications

Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster

Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster

Biodegradation of Aromatic Compounds by Escherichia coli

Characterization of a Novel Thermostable Mn(II)-dependent 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from a Polychlorinated Biphenyl- and Naphthalene-degrading Bacillus sp. JF8

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