Pinpointing Biphenyl Dioxygenase Residues That Are Crucial for Substrate Interaction

Zielinski, Marco; Kahl, Silke; Hecht, Hans‐Jürgen; Höfer, Bernd

doi:10.1128/jb.185.23.6976-6980.2003

Cited by 42 publications

(41 citation statements)

References 26 publications

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“…According to a model based on naphthalene dioxygenase crystal structure, it was recently demonstrated that BPDO residues that had not been predicted to be influential can play a major role in BPDO specificity (17). The present report provides a further example that the enzyme-substrate interactions that determine BPDO regiospecificity toward chlorobiphenyls are complex.…”

Section: Discussionmentioning

confidence: 74%

Revisiting the Regiospecificity of Burkholderia xenovorans LB400 Biphenyl Dioxygenase toward 2,2′-Dichlorobiphenyl and 2,3,2′,3′-Tetrachlorobiphenyl

Barriault

Lépine

Mohammadi

et al. 2004

Journal of Biological Chemistry

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2,2 -Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2 -chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2 ,3 -Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2 -CB and 2,3,2 ,3 -CB, thus providing new insights on the mechanism by which 2,2 -CB is dehalogenated to generate 2,3-dihydroxy-2 -chlorobiphenyl. We reacted 2,2 -CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2 -dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2 -CB-d 8 was determined to be a dihydroxychlorobiphenyl-d 7 by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2 ,3 -CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2 ,3 -tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2 -CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2 -CB and 2,3,2, ,3 -CB disfavors the dioxygenation of the chlorine-free orthometa carbons 5 and 6 for both congeners.The enzymes of the bacterial biphenyl catabolic pathway are very versatile. They can co-metabolically transform several polychlorinated biphenyls (1). The initial reaction of this pathway is catalyzed by the biphenyl dioxygenase (BPDO) 1 (2, 3). The cis- (2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene (commonly called cis-2,3-dihydro-2,3-dihydroxybiphenyl) generated by the catalytic oxygenation of biphenyl is dehydrogenated by the 2,3-dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (BphB) and the catechol produced is cleaved by the 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC). The 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid produced is then hydrolyzed by the 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid hydrolase to yield benzoate and 2-hydroxypentanoate (Fig. 1). The substrate specificity of BPDO is crucial, because it limits the range of compounds that can potentially be degraded by the catabolic system. BPDO is a three-component enzymatic system (4 -6) (Fig. 1). The first component is an iron-sulfur protein (ISP BPH ) that interacts with the substrate to catalyze the addition of molecular oxygen. The second and third components are a flavoprotein reductase and a ferredoxin that are involved in the transfer of electrons from N...

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Section: Discussionmentioning

confidence: 74%

Revisiting the Regiospecificity of Burkholderia xenovorans LB400 Biphenyl Dioxygenase toward 2,2′-Dichlorobiphenyl and 2,3,2′,3′-Tetrachlorobiphenyl

Barriault

Lépine

Mohammadi

et al. 2004

Journal of Biological Chemistry

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“…Several studies have reported that changes in amino acids that are not in the immediate vicinity of the bound substrate have a distinct effect on the structure of the active site. 15,16) The present results suggest that the higher affinities of 1072 Bph Dox for substrate are caused by the altered configuration of its binding pocket due to the indirect effect of amino acid replacement of His-255, Val-258, Gly-268, and Phe-277. It has also been reported that substrate binding induces significant conformational changes around the active site of RHA1 BphA1.…”

Section: Gln-226mentioning

confidence: 68%

Steady-State Kinetic Characterization of Evolved Biphenyl Dioxygenase, Which Acquired Novel Degradation Ability for Benzene and Toluene

Suenaga

Sato

Goto

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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“…Bruhlmann and Chen obtained some evolved BphDOs which could recognize both ortho-and para-substituted PCBs by DNA shuffling between LB400 and KF707 and found that all variants contained Thr335Ala and Phe336Ile substitutions (Gly335 and Ile336 in CumDO) (2). Zielinski et al reported that mutations at Met231, Phe378, and Phe384 (Met232, Phe378, and Tyr384 in CumDO) greatly altered the regiospecificity of substrate dioxygenation of BphDO LB400 (40). For NphDO, Phe352 (Phe378 in CumDO) has been shown to be a crucial residue for regio-and enantioselectivity (28).…”

Section: Resultsmentioning

confidence: 99%

“…Zielenski et al (40) and Suenaga et al (33) used the NphDO structure as the template for homology modeling of BphDO LB400 and BphDO KF707 and obtained fruitful results. However, some regions or residues which appear to be important for the substrate specificities of BphDOs but have not been thoroughly tested for engineering studies yet, such as Ile288 in loop 2, remain to be examined.…”

Section: Resultsmentioning

confidence: 99%

“…BphDOs of Burkhorderia cepacia LB400 (BphDO LB400) and Pseudomonas pseudoalcaligenes KF707 (BphDO KF707) have been extensively engineered to improve their capabilities for environmental pollutant degradation by using various techniques, such as random mutagenesis, in vitro DNA shuffling, and subunit or domain exchange (8,31,33,39,40). For members of the toluene/ biphenyl subfamily, however, crystallization of only one enzyme (BphDO from Burkhorderia sp.…”

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confidence: 99%

See 1 more Smart Citation

Crystal Structure of the Terminal Oxygenase Component of Cumene Dioxygenase fromPseudomonas fluorescensIP01

Dong¹,

Fushinobu²,

Fukuda³

et al. 2005

J Bacteriol

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The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 Å by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.Aromatic hydrocarbons are common contaminants of soil and groundwater (18). One of the most attractive means of removal of these compounds from the environment is the use of microorganisms (34). Dihydroxylation of the aromatic ring by a bacterial aromatic hydrocarbon dioxygenase is a prerequisite for subsequent oxidation of the aromatic nucleus by a ring fission dioxygenase (6). Aromatic hydrocarbon dioxygenases belong to a large family named the Rieske nonheme iron oxygenases (11). Werlen et al. delineated four dioxygenase subfamilies in this large family (the toluene/biphenyl, naphthalene, benzoate, and phthalate subfamilies) based on sequence alignment of the catalytic components (␣ subunits) (37). The toluene/biphenyl subfamily includes enzymes for the degradation of toluene, benzene, cumene (isopropylbenzene), biphenyl, and polychlorinated biphenyls (PCBs). The naphthalene subfamily consists of enzymes for the degradation of naphthalene and phenanthrene. The Rieske dioxygenases involved in bacterial hydrocarbon degradation comprise multicomponent enzyme systems (36) in which reduced pyridine nucleotide is used as the initial source of two electrons for dioxygen activation. The electrons pass through a flavin cofactor and Rieske [2Fe-2S] centers into the mononuclear iron center of the terminal Rieske nonheme iron dioxygenase component.The crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4 (NphDO) has been reported previously (4,15,17), and the structure-function relationship of this enzyme has been well studied (28). NphDO is an ␣ 3 ␤ 3 hexamer, and each ␣ subunit contains a Rieske [2Fe-2S] cluster and nonheme iron coordinated by His208, His213, and Asp362. The active site iron center of one of the ␣ subunits is directly connected by hydrogen bonds through a singl...

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Pinpointing Biphenyl Dioxygenase Residues That Are Crucial for Substrate Interaction

Cited by 42 publications

References 26 publications

Revisiting the Regiospecificity of Burkholderia xenovorans LB400 Biphenyl Dioxygenase toward 2,2′-Dichlorobiphenyl and 2,3,2′,3′-Tetrachlorobiphenyl

Revisiting the Regiospecificity of Burkholderia xenovorans LB400 Biphenyl Dioxygenase toward 2,2′-Dichlorobiphenyl and 2,3,2′,3′-Tetrachlorobiphenyl

Steady-State Kinetic Characterization of Evolved Biphenyl Dioxygenase, Which Acquired Novel Degradation Ability for Benzene and Toluene

Crystal Structure of the Terminal Oxygenase Component of Cumene Dioxygenase fromPseudomonas fluorescensIP01

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