Engineering a hybrid pseudomonad to acquire 3,4-dioxygenase activity for polychlorinated biphenyls

Suenaga, Hikaru; Nishi, Akito; Watanabe, Takahito; Sakai, Masashi; Furukawa, Koichi

doi:10.1016/s1389-1723(99)80090-5

Cited by 37 publications

(20 citation statements)

References 32 publications

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“…Other lines of experimental evidence are consistent with the assumption concerning the binding site in the CumDO structure. Kimura et al (19) and Suenaga et al (31,32) showed that a mutation at Thr376 (Thr377 in CumDO) in BphDO KF707 resulted in an ability to degrade PCB congeners. 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).…”

Section: Resultsmentioning

confidence: 99%

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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Section: Resultsmentioning

confidence: 99%

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

Dong¹,

Fushinobu²,

Fukuda³

et al. 2005

J Bacteriol

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“…We have previously reported that the four-amino-acid substitution in KF707 BphA1 permits great expansion of the ability to degrade single aromatic compounds such as benzene, toluene, and alkylbenzenes (37). From the structural information of KF707 BphA1, these four amino acids (His-255, Val-258, Gly-268, and Phe-227) are situated surrounding an active site (data not shown).…”

Section: Discussionmentioning

confidence: 98%

“…Previously we constructed a variety of chimeric bphA1 genes between KF707 and LB400 by using three common restriction sites (23). Then, we randomly recombined these two bphA1 genes by using DNA shuffling (24,37). Some chimeric dioxygenases possessed enhanced PCB-degrading abilities.…”

mentioning

confidence: 99%

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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“…Kimura et al (1997) constructed a variety of chimeric bphA1 genes by using three restriction nuclease sites common to the KF707-bphA1 and the LB400-bphA1 genes. Escherichia coli cells expressing BP Dox containing various chimeric BphA1 demonstrated that only a few amino acids in the carboxy-terminal half of BphA1 are involved in the recognition of the chlorinated ring and the sites of dioxygenation and thereby are responsible for the degradation of PCB (Kimura et al, 1997;Suenaga et al, 1999).…”

Section: Directed Evolution Of Biphenyl Dioxygenasesmentioning

confidence: 99%

“…These evolved bphA1s were successfully introduced into the bph gene cluster by recombination. One such strain, P. pseudoalcaligenes KF707-D34, retained the ability to degrade 4,4Ј-dichlorobiphenyl via 2,3-dioxygenation in a fashion identical to that of KF707 and gained a novel capability to degrade 2,5,4Ј-trichlorobiphenyl and 2,5,2Ј,5Ј-tetrachlorobiphenyl via 3,4-dioxygenation in a fashion identical to that of LB400 (Suenaga et al, 1999).…”

Section: Directed Evolution Of Biphenyl Dioxygenasesmentioning

confidence: 99%

Biochemical and genetic bases of microbial degradation of polychlorinated biphenyls(PCBs).

Furukawa

2000

J. Gen. Appl. Microbiol.

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105

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Engineering a hybrid pseudomonad to acquire 3,4-dioxygenase activity for polychlorinated biphenyls

Cited by 37 publications

References 32 publications

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

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

Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering

Biochemical and genetic bases of microbial degradation of polychlorinated biphenyls(PCBs).

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