Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering

Suenaga, Hikaru; Watanabe, Takahito; Sato, Mika; Ngadiman,; Furukawa, Koichi

doi:10.1128/jb.184.13.3682-3688.2002

Cited by 97 publications

(62 citation statements)

References 42 publications

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“…Supportive evidence provided by this work and a previous study (12) shows that residue 335 and especially residue 336 are directly implicated in enzyme turnover rates and regiospecificity. Replacing Phe 336 by Met or Ile as in p4 (Ala 335 -Met 336 ) or p8 (Ala 335 -Ile 336 ) variants resulted in a change in regiospecificity toward 2,2Ј-CB.…”

Section: Discussionsupporting

confidence: 59%

“…KF707 BPDO and B-356 BPDO catalyze the oxygenation of a much narrower range of PCB congeners than LB400 BPDO. Nevertheless, replacement of region III of LB400 BphA by that of KF707 or of B356 created enzymes that oxygenated a much broader range of PCB congeners (6,8,9,12). This shows that region III amino acids are not the only ones influencing the catalytic activity toward chlorobiphenyl, but it also shows that the sequence pattern of region III of LB400 BphA is not optimal for catalytic activity toward chlorobiphenyls.…”

mentioning

confidence: 97%

“…Thus Suenaga et al (12) found that replacing Ile 336 of KF707 BphA with Phe as in LB400 BphA changed the regiospecificity toward 2,2Ј-dichlorobiphenyl (2,2Ј-CB) to favor production of 2,3-dihydroxy-2Ј-chlorobiphenyl.…”

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confidence: 99%

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Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III

Barriault

Sylvestre

2004

Journal of Biological Chemistry

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It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) ␣ subunit are involved in substrate recognition and regiospecificity toward chlorobiphenyls. However, the sequence pattern of the amino acids of that segment of seven amino acids located in the C-terminal portion of the ␣ subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr 335 -Phe 336 -Ile 338 -Ile 341 ) of region III of Burkholderia xenovorans LB400 BphA. The library was screened for variants able to oxygenate 2,2 -dichlorobiphenyl (2,2 -CB). Replacement of Phe 336 with Met or Ile with a concomitant change of Thr 335 to Ala created new variants that transformed 2,2 -CB into 3,4-dihydro-3,4-dihydroxy-2,2 -dichlorobiphenyl, which is a dead end metabolite that was not cleaved by BphC. Replacement of Thr 335 -Phe 336 with Ala 335 -Leu 336 did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2 -CB that were oxygenated more efficiently by variant Ala 335 -Met 336 than by LB400 BPDO was similar for both enzymes. Thus structural changes that altered the regiospecificity toward 2,2 -CB did not affect the metabolite profile of other congeners, although it affected the rate of conversion of these congeners. It was especially noteworthy that both LB400 BPDO and the Ala 335 -Met 336 variant generated 2,3-dihydroxy-2 ,4,4 -trichlorobiphenyl as the sole metabolite from 2,4,2 ,4 -CB and 4,5-dihydro-4,5-dihydroxy-2,3,2 ,3 -tetrachlorobiphenyl as the major metabolite from 2,3,2 ,3 -CB. This shows that 2,4,2 ,4 -CB is oxygenated principally onto vicinal ortho-meta carbons 2 and 3 and that 2,3,2 ,3 -CB is oxygenated onto meta-para carbons 4 and 5 by both enzymes. The data suggest that interactions between the chlorine substitutes on the phenyl ring and specific amino acid residues of the protein influence the orientation of the phenyl ring inside the catalytic pocket.Biphenyl dioxygenase (BPDO) 1 catalyzes the first reaction of the biphenyl catabolic pathway. BPDO comprises three components (1, 2): The iron-sulfur oxygenase (ISP BPH ) made up of an ␣ subunit (M r ϭ 51,000) and a ␤ subunit (M r ϭ 22,000), the ferredoxin (FER BPH , M r ϭ 12,000), and the ferredoxin reductase (RED BPH , M r ϭ 43,000). The encoding genes for Burkholderia xenovorans LB400 (3), which is also called Burkholderia (Pseudomonas) sp. LB400 (4, 5), are bphA (ISP BPH ␣ subunit), bphE (ISP BPH ␤ subunit), bphF (FER BPH ), and bphG (RED BPH ). BPDO can oxygenate several polychlorinated biphenyl (PCB) congeners. The application of BPDO for efficient PCB degrading processes will require an expansion of the range of PCB substrates it can oxygenate. The catalytic oxygenation of biphenyl occurs normally on carbons 2 and 3 to generate the cis-2,3-dihydro-2,3-dihydroxybiphenyl, which is dehydrogenated by the cis-2,3-dihydro-2,3-dihydroxybiphenyl 2,3-dehydrogenase encoded by bphB in strain LB400. The resulting catechol 2,3-dihydroxybiphenyl is then cleaved b...

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Section: Discussionsupporting

confidence: 59%

mentioning

confidence: 97%

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Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III

Barriault

Sylvestre

2004

Journal of Biological Chemistry

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“…[11c, 12] Concerning BPDO, residues Ile335 and Thr376 were determined to alter the substrate scope and regioselectivity for the dihydroxylation of biphenyl. [13] Previous studies using Pseudomonas pseudoalcaligenes KF707 BPDO, demonstrated that Ile335 was very important for determining substrate specificity and regioselectivity. The substrate scope for a range of polychlorinated biphenyls (PCB) was strongly associated with the amino acid side chain in this position.…”

Section: Mutant's Selection and Designmentioning

confidence: 99%

“…All enzymes with broad substrate specificity contain asparagine at the 376 position, whereas the enzymes with narrow substrate specificity contain threonine at this site. [15] Different mutations have been performed on this residue of BPDO. The introduction of amino acids smaller than Thr resulted in enzyme variants with reduced activity, while the inclusion of larger amino acids precluded substrate binding.…”

Section: Mutant's Selection and Designmentioning

confidence: 99%

Site‐Directed Mutagenesis Studies on the Toluene Dioxygenase Enzymatic System: Role of Phenylalanine 366, Threonine 365 and Isoleucine 324 in the Chemo‐, Regio‐, and Stereoselectivity

et al. 2017

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Toluene Dioxygenase (TDO) enzymatic complex has been widely used as a biocatalyst for the regio-and enantioselective preparation of ciscyclohexadienediols, which are very important starting materials for organic synthesis. However, the lack of regio-and stereodiversity of the dioxygenation process by TDO and related dioxygenases constitutes a clear drawback when planning the use of these diols in synthetic schemes. In this work, we developed three TDO mutants in residues phenylalanine 366, threonine 365 and isoleucine 324, with the aim to alter the chemo-, regio-and stereoselectivity of the biotransformation of arenes. While no changes in the regioselectivity of the process were observed, dramatic variations in the chemo-and enantioselectivity were found for mutants I324F, T365N and F366 V in a substratedependent manner.

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Application of Aromatic Hydrocarbon Dioxygenases

Tan

Parales

2016

Green Biocatalysis

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c h a p t e r 1 7 INTRODUCTIONThe first reported aromatic hydrocarbon dioxygenase, toluene dioxygenase, was identified to catalyze cis-dihydroxylation of benzene and toluene in Pseudomonas putida F1 [1, 2]. Since then, many more dioxygenases have been studied, and the crys tal structures of several have been determined [3]. This group of enzymes plays an important role in the initial step in the biodegradation of both naturally occurring and man-made aromatic compounds, including aromatic acids, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and nitroaromatic, aminoaromatic and halogenated aromatic compounds [4,5]. Dioxygenases are nonheme Rieske-type NAD(P)H-dependent enzymes that intro duce both atoms of molecular oxygen into their substrates. The multicomponent enzyme systems are composed of a catalytic oxygenase component and one-or two-electron trans fer proteins, including a flavoprotein reductase, and in some cases a ferredoxin that medi ates electron transfer from the reductase to the oxygenase component ([3]; Figure 17.1).In general, Rieske dioxygenases are capable of oxidizing a broad range of substrates, well beyond the range of compounds that serves as growth substrates for the host bacterium [4,5,7]. For example, toluene dioxygenase from P. putida F1 facilitates the oxidation of over 200 different substrates [7], and naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4 has been documented to oxidize over 60 different substrates [8]. In addition, these enzymes catalyze diverse types of reactions, including cisdihydroxylations, O-and N-dealkylations, desaturations, and the formation of chiral sulfoxides from sulfides ([7-9]; Figure 17.2). In many cases the products are chiral com pounds that are high in enantiomeric purity [8,10,11]. For the aforementioned reasons, dioxygenases are attractive biocatalysts for bioremediation and industrial applications. CHALLENGES IN AROMATIC HYDROCARBON DIOXYGENASE APPLICATIONSThe practical use of dioxygenases in industry is generally limited to the use of whole-cell biocatalysts due to several challenges. The catalytic activity of dioxygenases is dependent on reducing equivalents provided by NADH or NADPH, which require regeneration by electron transfer proteins [12,13]. The regeneration process is typically a limiting step

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Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering

Cited by 97 publications

References 42 publications

Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III

Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III

Site‐Directed Mutagenesis Studies on the Toluene Dioxygenase Enzymatic System: Role of Phenylalanine 366, Threonine 365 and Isoleucine 324 in the Chemo‐, Regio‐, and Stereoselectivity

Application of Aromatic Hydrocarbon Dioxygenases

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