Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase

Tetsuya, Kumamaru; Suenaga, Hikaru; Mitsuoka, Muneshi; Watanabe, Takahito; Furukawa, Koichi

doi:10.1038/nbt0798-663

Cited by 217 publications

(96 citation statements)

References 33 publications

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“…For instance, the D-selectivity of a native hydan-toinase was reversed by mutagenesis and selective screening procedures, giving an L-specific enzyme with no equivalent in nature, able to catalyze the production of L-methionine 26 . Gene-shuffling techniques have been used to randomly recombine genes from two organisms possessing biphenyl dioxygenase activity, yielding a recombinant enzyme capable of oxidative degradation of polychlori-nated biphenyls (PCBs) and simple aromatic monomers 27 . Molecular stability.…”

Section: Product Recoverymentioning

confidence: 99%

The search for the ideal biocatalyst

2002

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While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.The past two decades have led to major advances in our understanding of the subtleties of protein structure-function interrelationships. Mechanisms of protein stability in aqueous and nonaqueous environments 1,2 , the links between conformational mobility, structural integrity and activity 3,4 , and the complexities of substrate specificity have all succumbed to the onslaught of advanced molecular methods, including crystallography 5-7 , site-specific mutagenesis, gene shuffling, and protein evolution [8][9][10][11] . Scientists are now in a position to visualize, if not design, catalytic systems that approach the functional "ideal".The "ideal" catalyst is typically considered by the biochemist in terms of turnover number (k cat ) or, for a given process, in terms of maximum specificity constant (A cat /KM). However, from a bioprocess viewpoint, each bioprocess is constrained by a set of conditions dictated by the specific properties of the substrates, products, and the bioconversion reaction. Thus, while for any given bioprocess it is clearly possible to specify a set of catalyst properties that would constitute the ideal for that process, only broad generalizations for ideality may otherwise be identified.In this review, we discuss molecular properties of enzymes from a bioprocess viewpoint. A paradigm for design based on the ideal process determining the desired features of the catalyst is presented. "Ideal" characteristics (generic and process-specific) and methodologies for seeking or engineering these are discussed. We then review the extent to which significant changes in protein functional properties have been achieved by application of these methods, together with issues that require more research and/or development. Ideal processesBioprocess engineers develop processes not only against fixed capital and operating expenditure, but also rapid, and often, very aggressive timelines. This is particularly the case for pharmaceutical products where patent expiry on the product demands a robust and scalable process with short development times 12 . Conventionally, once a synthetic route is fixed and the biocatalytic step defined, bioprocess design and operation are centered on the properties of the

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Section: Product Recoverymentioning

confidence: 99%

The search for the ideal biocatalyst

2002

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“…After 18 h of incubation at 37°C, the membranes were transferred to fresh LB plates containing 1 mM isopropyl ␤-Dthiogalactopyranoside (IPTG), the plates were incubated for 3 h at 37°C, and then crystals of 4-CB or of 2,2Ј-CB were placed into the lid of the Petri dishes. 4-CB is a chlorobiphenyl congener that is normally oxidized at rate similar or higher than biphenyl by most BPDOs of natural occurrence, including LB400 BPDO (8). It was therefore used to determine the proportion of the clones of the library expressing an enzyme for which the mutations that occurred in region III did not alter their capacity to oxygenate this easily oxidizable chlorobiphenyl.…”

Section: Mutagenesis At Targeted Multi-sites Of Bphamentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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“…Previously, we constructed various chimeric bphA1 variants by using DNA shuffling between KF707 and LB400 bphA1 genes. 7) Screening of these clones allowed us to obtain evolved Bph Dox, which exhibits enhanced abilities to degrade certain PCBs and biphenyl-related compounds. In particular, it is noteworthy that an Escherichia coli clone carrying pSHF1072 plasmid exhibits wide and enhanced capabilities to degrade not only alkylbenzenes but also benzene and toluene, compounds that are rarely attacked by wild-type KF707 Bph Dox.…”

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confidence: 99%