Characterization of an Escherichia coli aromatic hydroxylase with a broad substrate range

Prieto, María Auxiliadora; Pérez-Aranda, A.; García, José Luis Rueda

doi:10.1128/jb.175.7.2162-2167.1993

Cited by 66 publications

(71 citation statements)

References 37 publications

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“…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 (38,39). In spite of this information, some genes of the 4-HPA catabolic pathway as well as their genetic arrangement remained unknown.…”

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confidence: 99%

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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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“…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).…”

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

1996

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“…This monooxygenase shows a broad substrate range, hydroxylating phenol derivatives (32,33). While the HpaB protein (58.7 kDa) of 4-HPA 3-monooxygenase was shown to be the oxygenase component, HpaC (18.6 kDa) was assumed to be a coupling protein that enhanced the activity of HpaB and could prevent the wasteful oxidation of NADH in the absence of substrate (32).…”

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“…The 4-hydroxyphenylacetate (4-HPA) 3-monooxygenase from Escherichia coli W is a two-component enzyme encoded by the hpaB and hpaC genes and catalyzes the initial reaction in the degradation of 4-HPA, i.e., the introduction of a second hydroxyl group into the benzene nucleus at a position ortho to the existing hydroxyl group, giving rise to 3,4-dihydroxyphenylacetate (3,4-DHPA) (32,33). This monooxygenase shows a broad substrate range, hydroxylating phenol derivatives (32,33).…”

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Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase of Escherichia coli W: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

et al. 2000

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“…putida, R. pickettii, and Burkholderia cepacia (Prieto et al, 1993;Prieto and Garcia, 1994;Amnachalam et al, 1992Amnachalam et al, , 1994Arunachalam and Massay, 1994;Takizawa et al, 199.5;Xun, 1996). From the data compiled in Table 3, a great variety within this class of enzymes becomes obvious.…”

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Two‐Component Flavin‐dependent Pyrrole‐2‐carboxylate Monooxygenase from Rhodococcus Sp.

Becker

Schräder

Andreesen

1997

European Journal of Biochemistry

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Pyrrole-2-carboxylate can serve as the sole source of carbon, nitrogen, and energy for a strain tentatively identified to belong to the genus Rhodococcus. An NADH-dependent oxygenase activity was detected in cell extracts that initiated the degradation of the substrate. During purification of the enzyme, this activity was separated into two protein components which were both purified to apparent homogeneity. A small monomeric 18.7-kDa protein designated as reductase, catalyzed in vitro the NADH and FAD-dependent reduction of cytochrome c and had an NADH-oxidase activity. The second component, a 54-kDa protein with a trimeric native structure had no enzymatic activity by itself, but exhibited a pyrrole-2-carboxylate-dependent oxygen consumption when it was complemented with the reductase component, FAD, and NADH. This indicated that the large protein referred to as oxygenase was responsible for the oxygen-dependent hydroxylation of the substrate. The rate of an uncoupled NADH oxidation without hydroxylation of the substrate was found to be strongly dependent on the molar ratio of both components. The uncoupling was nearly completely suppressed by a 5-7-fold molar excess of the oxygenase component. The small protein was N-terminally blocked. It was thus proteolytically digested and four of the resulting peptides were sequenced comprising 47 amino acids. The sequences of these fragments were similar to the sequences reported for the small component of different two-component flavin monooxygenases. Furthermore, the N-terminus of the oxygenase component showed high sequence similarity to the second, usually large subunit of these enzymes and to two single-component flavin monooxygenases. Thus, the enzyme from Rhodococcus sp. designated as pyrrole-2-carboxylate monooxygenase belongs to the recently discovered new class of two-component flavin aromatic monooxygenases. Some of the basic properties of both components were determined and their interaction during catalysis was investigated.

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Characterization of an Escherichia coli aromatic hydroxylase with a broad substrate range

Abstract: The hpaB gene encoding an aromatic hydroxylase of Escherichia coli ATCC 11105, a penicillin G acylase-producing strain, has been cloned and expressed in E. coli K-12

Cited by 66 publications

References 37 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

Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase of Escherichia coli W: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

Two‐Component Flavin‐dependent Pyrrole‐2‐carboxylate Monooxygenase from Rhodococcus Sp.

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