Tuning biphenyl dioxygenase for extended substrate specificity

Brühlmann, Fredi; Chen, Wilfred

doi:10.1002/(sici)1097-0290(19990605)63:5<544::aid-bit4>3.0.co;2-6

Cited by 90 publications

(43 citation statements)

References 21 publications

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“…These results clearly indicate that Ile-335 in KF707 Bph Dox plays a crucial role in the degradation of 4,4Ј-dichlorobiphenyl. This is in agreement with the report by Brühlmann and Chen that substitutions Thr335Ala and Phe336Ile in LB400 BphA1 are important in substrate specificity (5).…”

Section: Discussionsupporting

confidence: 93%

Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering

et al. 2002

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Structure 6:571-586, 1998), we developed a three-dimensional model of KF707 BphA1 of Pseudomonas pseudoalcaligenes KF707. Based on structural information about the amino acids which coordinate the catalytic nonheme iron center, we constructed 12 site-directed BphA1 mutants with changes at positions 227, 332, 335, 376, 377, and 383 and expressed these enzymes in Escherichia coli. The Ile335Phe, Thr376Asn, and Phe377Leu Bph Dox mutants exhibited altered regiospecificities for various PCBs compared with wild-type Bph Dox. In particular, the Ile335Phe mutant acquired the ability to degrade 2,5,2,5-tetrachlorobiphenyl by 3,4-dioxygenation and showed bifunctional 2,3-dioxygenase and 3,4-dioxygenase activities for 2,5,2-trichlorobiphenyl and 2,5,4-trichlorobiphenyl. Furthermore, two mutants, the Phe227Val and Phe377Ala mutants, introduced molecular oxygen at the 2,3 position, forming 3-chloro-2,3-dihydroxy biphenyl with concomitant dechlorination.

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Section: Discussionsupporting

confidence: 93%

Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering

et al. 2002

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“…3). Carbon resonances were assigned based on the simulated 13 C spectra of possible 4-Cl and 4-OH HOPDA tautomers and are consistent with the tautomers identified by 1 H NMR discussed above. In 4-Cl HOPDA, H-5 A showed a cross-peak with carbonyl C-6 A (189 ppm), which in turn showed a cross-peak with the aromatic ortho H A values.…”

Section: Specificity Of a Pcb-degrading Hydrolasesupporting

confidence: 64%

Identification of a Serine Hydrolase as a Key Determinant in the Microbial Degradation of Polychlorinated Biphenyls

Seah¹,

Labbé²,

Nerdinger³

et al. 2000

Journal of Biological Chemistry

100

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The ability of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) of Burkholderia cepacia LB400 to hydrolyze polychlorinated biphenyl (PCB) metabolites was assessed by determining its specificity for monochlorinated HOPDAs. The relative specificities of BphD for HOPDAs bearing chlorine substituents on the phenyl moiety were 0.28, 0.38, and 1.1 for 8-Cl, 9-Cl, and 10-Cl HOPDA, respectively, versus HOPDA (100 mM phosphate, pH 7.5, 25°C). In contrast, HOPDAs bearing chlorine substituents on the dienoate moiety were poor substrates for BphD, which hydro-

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“…Sitedirected and random mutagenesis studies with biphenyl dioxygenases from Burkholderia sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707 have identified amino acids in the oxygenase ␣ subunits that are important in determining PCB congener specificity (9,17,36,39,45). In particular, replacement of Asn-377 by Thr in BPDO LB400 significantly extended the range of PCBs oxidized (45).…”

Section: Discussionmentioning

confidence: 99%

Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme

et al. 2000

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The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio-and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. The naphthalene dioxygenase (NDO) enzyme system (EC 1.14.12.12) from Pseudomonas sp. strain NCIB 9816-4 catalyzes the first step in the aerobic degradation of naphthalene. In this reaction ( Fig. 1), NDO adds both atoms of oxygen to the aromatic nucleus of naphthalene, forming homochiral (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (30, 31). In addition, NDO catalyzes the oxidation of a wide variety of aromatic compounds to enantiomerically pure chiral products (8, 56). The NDO system consists of three components, each of which has been purified and characterized. An iron-sulfur flavoprotein reductase and an iron-sulfur ferredoxin transfer electrons from NAD(P)H to the catalytic oxygenase component (14,15,23,24). The oxygenase is composed of large and small subunits, ␣ and ␤, respectively, that are in an ␣ 3 ␤ 3 configuration (35). NDO is a member of a large family of oxygenases whose ␣ subunits contain a Rieske [2Fe-2S] center and mononuclear nonheme iron (10). In the NDO system, electrons are transferred from the Rieske center of the ferredoxin to the Rieske center of the oxygenase ␣ subunit. The reduced Rieske center in one ␣ subunit transfers an electron to mononuclear iron at the active site in an adjacent ␣ subunit (35, 50). His-208, His-213, and Asp-362 coordinate the active-site iron, forming a 2-His-1-carboxylate facial triad. This structural motif is found in other mononuclear nonheme iron enzymes, including tyrosine hydroxylase, isopenicillin synthetase, and 2,3-dihydroxybiphenyl 1,2-dioxygenase (26, 40). Asp-205 in the catalytic domain of the NDO ␣ subunit is hydrogen bonded to His-208 and to His-104 in the adjacent ␣ subunit (Fig. 2). His-104 is one of the Rieske center ligands. Asp-205 has been shown to be required for efficient electron transfer from the Rieske center to the active-site iron (50).Recent studies have shown that the oxygenase ␣ subunits are responsible for determining the substrate specificities of NDO and the related enzymes 2-nitrotoluene dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 and 2,4-dinitrotoluene dioxygenase (DNTDO) from Burkholderia sp. strain DNT (48...

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Tuning biphenyl dioxygenase for extended substrate specificity

Cited by 90 publications

References 21 publications

Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering

Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering

Identification of a Serine Hydrolase as a Key Determinant in the Microbial Degradation of Polychlorinated Biphenyls

Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme

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