Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding 1 1Edited by D. C. Rees

Carredano, Enrique; Karlsson, Andreas; Kauppi, Björn; Choudhury, Devapriya; Parales, Rebecca E.; Parales, J.V.; Lee, Kyung Ho; Gibson, David T.; Eklund, Hans; Ramaswamy, S.

doi:10.1006/jmbi.1999.3462

Cited by 155 publications

(191 citation statements)

References 30 publications

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“…If only a fraction of the amino acid residues that determine a given property are exchanged, context dependence of the effects of such replacements is inevitable. This and other publications (Beil et al, 1998 ;Kauppi et al, 1998 ;Carredano et al, 2000 ;Parales et al, 2000) suggest that the structure of the substrate-binding site is determined by segments that are rather distant in the sequence. Thus, context dependence is expected for the effects of individual exchanges of such segments on the structure of the binding site.…”

Section: Discussionsupporting

confidence: 60%

“…The corresponding region of BDO-P6 differs in 70 (42 %) of the residues. Thus, the highly similar regiospecificity of dioxygenation by BDO-H04 and BDO-P6 makes it unlikely that region αA significantly participates in formation of the substrate-binding pocket, although an α subunit sequence alignment with a naphthalene dioxygenase, for which three-dimensional crystal structures have been determined (Kauppi et al, 1998 ;Carredano et al, 2000), suggests that the Nterminal residues up to about position 55 are part of the ' catalytic domain ' (Fig. 2) that harbours the active site.…”

Section: Discussionmentioning

confidence: 99%

“…Electrophoresis was carried out in 0n1 % SDS\12 % polyacrylamide gels as described previously (Hofer et al, 1993). Gels were stained with Coomassie brilliant blue R250 (Sambrook et al, 1989 deduced from the crystal structure of a naphthalene dioxygenase (Kauppi et al, 1998 ;Carredano et al, 2000) and from functional studies on BDO variants (Erickson & Mondello, 1993 ;Kimura et al, 1997 ;Mondello et al, 1997). Hatched and white boxes represent the catalytic and Rieske domains, respectively.…”

Section: Methodsmentioning

confidence: 99%

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The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

2002

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Protein engineering by segment exchange was used to distinguish between regions of major and minor influence on the structure of the substrate-binding pocket of a biphenyl dioxygenase (BDO). Eight chimaeric enzyme systems were generated that each consisted of a hybrid hydroxylase α subunit (BphA1) containing segments from Burkholderia sp. strain LB400 and Rhodococcus globerulus P6, and of a hydroxylase β subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400. All hybrid bphA1 genes were expressed at high levels. Seven of the resulting fusion subunits functionally interacted with the other polypeptides of the dioxygenase system to yield catalytically active enzymes. Changes in the regiospecificity of substrate attack, monitored by the formation of seventeen different dioxygenation products obtained from seven chlorobiphenyls, were used to monitor effects of segment exchanges on the structure of the BDO substratebinding site. Exchanges of neither the β subunit nor the N-and C-terminal regions of the α subunit exerted significant influences. All BDO regions that showed major effects on the substrate-binding pocket were located between approximately positions 165 and 395 of the α subunit. Within this part of the enzyme, in addition to segments identified previously, a subregion which is involved in ligation of the mononuclear iron significantly influenced the regiospecificity of substrate dioxygenation. Moreover, the results indicate that the construction of appropriate hybrid genes may be used as a general strategy to overcome problems in obtaining heterologous BDO activities in Escherichia coli or other host organisms.

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Section: Discussionsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

2002

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“…These segments encode the major determinants of substrate and product spectra, according to biochemical studies with enzyme variants (Erickson & Mondello, 1993;Kimura et al, 1997;Mondello et al, 1997;Beil et al, 1998;Zielinski et al, 2002). They also encode all amino acid residues that, by analogy with the three-dimensional crystal structure of a related naphthalene dioxygenase (Carredano et al, 2000), form the substrate-binding pocket (see also Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

A genetic system for the rapid isolation of aromatic-ring-hydroxylating dioxygenase activities

Kahl

Höfer

2003

Microbiology

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Aromatic-ring-hydroxylating dioxygenases (ARHDOs) are key enzymes in the aerobic bacterial metabolism of aromatic compounds. They are of biotechnological importance as they function as biocatalysts in the stereospecific synthesis of chiral synthons and the degradation of aromatic pollutants. This report describes the development and validation of a system for the rapid isolation and characterization of specific ARHDO activities. The system is based on the identification of ARHDO gene segments that encode the enzymes' major functional determinants, on consensus primers for the direct amplification of such partial genes and on a 'recipient' ARHDO gene cluster for the insertion of the amplified segments. Previously, it has been shown that neither the N-nor the C-terminal portions but only the core region of the large or a-subunit of a class II ARHDO significantly influence substrate and product spectra. On the basis of these observations, consensus primers were designed for the amplification of the gene segment encoding the catalytic core of the large subunit. These primers were tested on 11 bacterial isolates known to metabolize aromatic compounds. In 10 cases, a gene fragment of expected length was amplified. DNA sequencing confirmed similarity to ARHDO a-subunit gene cores. The heterologously well-expressible bphA gene cluster of Burkholderia sp. strain LB400 was modified to facilitate the in-frame insertion of amplified segments. It was used successfully to express the resulting hybrid gene clusters and to form catalytically active chimaeric ARHDOs. The metabolic properties of these enzymes differed significantly from each other and from the parental ARHDO of strain LB400. These results indicate that the system described here can be used to rapidly isolate and functionally characterize ARHDO activities, starting from isolated strains, mixtures of organisms or samples of nucleic acids. Applications of the system range from the recruitment of novel ARHDO activities to an improved characterization of natural ARHDO diversity.

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“…X-ray crystallographic studies show that the substrate binds near the mononuclear iron center, and thus, this is the likely site of oxygen activation and insertion. 18 …”

Section: Rieske Cis-dihydrodiol Forming Dioxygenases Overview Of the mentioning

confidence: 99%

Finding Intermediates in the O₂ Activation Pathways of Non-Heme Iron Oxygenases

et al. 2007

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Intermediates in the reaction cycle of an oxygenase are usually very informative of the chemical mechanism of O 2 activation and insertion. However, detection of these intermediates is often complicated by their short lifetime and the regulatory mechanism of the enzyme designed to ensure specificity. Here, the methods used to detect the intermediates in an extradiol dioxygenase, a Rieske cis-dihydrodiol dioxygenase, and soluble methane monooxygenase are discussed. The methods include the use of alternative, chromophoric substrates, mutagenesis of active site catalytic residues, forced changes in substrate binding order, control of reaction rates using regulatory proteins, and initialization of catalysis in crystallo.

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Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding 1 1Edited by D. C. Rees

Cited by 155 publications

References 30 publications

The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

A genetic system for the rapid isolation of aromatic-ring-hydroxylating dioxygenase activities

Finding Intermediates in the O₂ Activation Pathways of Non-Heme Iron Oxygenases

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Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding 1 1Edited by D. C. Rees

Cited by 155 publications

References 30 publications

The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

A genetic system for the rapid isolation of aromatic-ring-hydroxylating dioxygenase activities

Finding Intermediates in the O2 Activation Pathways of Non-Heme Iron Oxygenases

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Finding Intermediates in the O₂ Activation Pathways of Non-Heme Iron Oxygenases