Cloning, characterization, and sequence analysis of the clcE gene encoding the maleylacetate reductase of Pseudomonas sp. strain B13

Kasberg, T; Seibert, Volker; Schlömann, Michael; Reineke, Walter

doi:10.1128/jb.179.11.3801-3803.1997

Cited by 23 publications

(20 citation statements)

References 17 publications

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“…Although the maleylacetate reductase gene, tfdF, of Ralstonia eutropha JMP134(pJP4) was initially misinterpreted as encoding a chlorodienelactone isomerase (Don et al, 1985), the chlorocatechol gene clusters of pJP4, pAC27, Pseudomonas sp. B13, and pP51 by DNA sequencing, by correspondence of experimentally determined N-terminal sequences to sequences predicted from DNA and by expression of cloned genes, were shown to contain maleylacetate reductase genes in addition to genes for chlorocatechol 1,2-dioxygenases, chloromuconate cycloisomerases and dienelactone hydrolases (Frantz & Chakrabarty, 1987;Perkins et al, 1990;Laemmli et al, 2000;van der Meer et al, 1991;Schell et al, 1994;Kasberg et al, 1995Kasberg et al, , 1997Seibert et al, 1993;Plumeier et al, 2002). Sequence similarities suggest that this is true also for the chlorocatechol gene clusters of various other plasmids or strains.…”

Section: Introductionmentioning

confidence: 99%

“…The outgroups were removed afterwards and resulting column gaps were eliminated. References for the published sequences (order as in the alignment): AF130250 (this study); U19883 ; AF169302 (Johnson et al, 2002); NZ_AAAI01000075, Ralstonia metallidurans Reut_75 genome shotgun sequence; AF512952 (Cai & Xun, 2002); NC_003304, positions 2 501 013-2 502 068, Agrobacterium tumefaciens C58 chromosome (Wood et al, 2001); NC_004463, positions 2 938 247-2 939 326, Bradyrhizobium japonicum USDA 110 genome (Kaneko et al, 2002); AJ536297 (Fritsche, 1998); X72850 (Armengaud et al, 1999); U32188 (Vedler et al, 2000); AF030176 (Seibert et al, 1998);M57629 (van der Meer et al, 1991); AF019038 (Kasberg et al, 1997); M35097 (Perkins et al, 1990); AB050198 (Liu et al, 2001); P94135 (Laemmli et al, 2000); NC_004369, positions 3 077 929-3 079 059, Corynebacterium efficiens YS-314 T genome; NC_003450, positions 1 214 871-1 215 941 and 3 257 401-3 258 492, Corynebacterium glutamicum ATCC 13032 T genome; NC_004463, positions 1 096 165-1 097 388, Bradyrhizobium japonicum USDA 110 genome (Kaneko et al, 2002). Fig.…”

Section: Preliminary Characterization Of Orfs Neighbouring the Maleylmentioning

confidence: 99%

“…B13, and pP51 by DNA sequencing, by correspondence of experimentally determined N-terminal sequences to sequences predicted from DNA and by expression of cloned genes, were shown to contain maleylacetate reductase genes in addition to genes for chlorocatechol 1,2-dioxygenases, chloromuconate cycloisomerases and dienelactone hydrolases (Frantz & Chakrabarty, 1987;Perkins et al, 1990;Laemmli et al, 2000;van der Meer et al, 1991;Schell et al, 1994;Kasberg et al, 1995Kasberg et al, , 1997Seibert et al, 1993;Plumeier et al, 2002 During growth with 4-fluorobenzoate, R. eutropha 335 T and R. eutropha JMP222, a pJP4-free derivative of the 2,4-dichlorophenoxyacetate-utilizing strain JMP134, induce a maleylacetate reductase, but not the other enzymes typical for chlorocatechol catabolism (Schlömann et al, 1990a). Thus, these strains contain a presumably chromosomal maleylacetate reductase gene of still unknown metabolic affiliation.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate

et al. 2004

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A gene cluster containing a gene for maleylacetate reductase (EC 1.3.1.32) was cloned from Ralstonia eutropha 335 T (DSM 531 T ), which is able to utilize 4-fluorobenzoate as sole carbon source. Sequencing of this gene cluster showed that the R. eutropha 335 T maleylacetate reductase gene, macA, is part of a novel gene cluster, which is not related to the known maleylacetate-reductase-encoding gene clusters. It otherwise comprises a gene for a hypothetical membrane transport protein, macB, possibly co-transcribed with macA, and a presumed regulatory gene, macR, which is divergently transcribed from macBA. MacA was found to be most closely related to TftE, the maleylacetate reductase from Burkholderia cepacia AC1100 (62 % identical positions) and to a presumed maleylacetate reductase from a dinitrotoluene catabolic gene cluster from B. cepacia R34 (61 % identical positions). By expressing macA in Escherichia coli, it was confirmed that macA encodes a functional maleylacetate reductase. Purification of maleylacetate reductase from 4-fluorobenzoate-grown R. eutropha 335 T cells allowed determination of the N-terminal sequence of the purified protein, which was shown to be identical to that predicted from the cloned macA gene, thus proving that the gene is, in fact, recruited for growth of R. eutropha 335 T with this substrate. INTRODUCTIONMaleylacetate reductases (EC 1.3.1.32) play a crucial role in the aerobic microbial degradation of aromatic compounds. They catalyse the NADH-or NADPH-dependent reduction of maleylacetate or 2-chloromaleylacetate to 3-oxoadipate ( Fig. 1) or of substituted maleylacetates to substituted 3-oxoadipates. In fungi, maleylacetate reductases contribute to the catabolism of very common substrates, such as tyrosine, gentisate, benzoate, 4-hydroxybenzoate, protocatechuate, vanillate, resorcinol and phenol (Karasevich & Ivoilov, 1977;Buswell & Eriksson, 1979;Gaal & Neujahr, 1979;Sparnins et al., 1979;Anderson & Dagley, 1980;Jones et al., 1995). For bacteria, it has been shown that maleylacetate reductases are involved in the degradation of resorcinol and 2,4-dihydroxybenzoate via hydroxyquinol (Larway & Evans, 1965;Chapman & Ribbons, 1976;Stolz & Knackmuss, 1993). Other substrates whose catabolic routes comprise this activity have a more complex structure, such as 2-hydroxydibenzo-p-dioxin and 3-hydroxydibenzofuran (Armengaud et al., 1999), or carry unusual substituents, such as nitro or sulfo groups (Spain & Gibson, 1991;Feigel & Knackmuss, 1993;Jain et al., 1994;Rani & Lalithakumari, 1994), fluorine or chlorine atoms (Duxbury et al., 1970; Schlömann et al., 1990a;Latus et al., 1995;Zaborina et al., 1995;Daubaras et al., 1996;Miyauchi et al., 1999).The maleylacetate reductase genes characterized so far tend to belong to specialized gene clusters for the degradation of aromatic compounds. Thus, 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin degradation via hydroxyquinol appears to be encoded by a specialized gene cluster comprising most of the genes for the pathway including a maleylac...

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Section: Introductionmentioning

confidence: 99%

Section: Preliminary Characterization Of Orfs Neighbouring the Maleylmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate

et al. 2004

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“…Finally, 3-chlorocatechol is degraded to ␤-ketoadipate by the so called modified ortho pathway (clc, chromosome 1), organized similar to Pseudomonas sp. B13 (42).…”

Section: Genetic Factors Indicating the Ecological Niche Of B Xenovomentioning

confidence: 99%

Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

Chain

Denef

Konstantinidis

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

325

280

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Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven ''central aromatic'' and twenty ''peripheral aromatic'' pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.genomics ͉ niche adaptation ͉ evolution ͉ biodegradation ͉ redundancy

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“…The clcE gene encoding maleylacetate reductase is immediately after the clcD gene encoding DLH; consequently there is a ribosome binding site and suspected proteolytic cleavage site at the end of the DLH gene. 25 We suspected that the nucleotides encoding the three C-terminal DLH residues are inconsequential to the function of DLH but are necessary for translation of maleylacetate reductase. Deletion of the three C-terminal residues, as in the S-Δ variant, did in fact improve the stability and solubility (Supporting Figure S2, Supporting Information) of the enzyme lending credence to this hypothesis.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Directed Evolution of New and Improved Enzyme Functions Using an Evolutionary Intermediate and Multidirectional Search

et al. 2014

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ABSTRACT:The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.A lthough enzymes are remarkable catalysts, their use in many practical applications is limited by the availability of large quantities of stable enzyme with appropriate catalytic properties. Directed evolution can be usefully employed to enhance enzyme stability and alter substrate specificity to engineer tailored biocatalysts.

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Cloning, characterization, and sequence analysis of the clcE gene encoding the maleylacetate reductase of Pseudomonas sp. strain B13

Cited by 23 publications

References 17 publications

Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate

Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate

Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

Directed Evolution of New and Improved Enzyme Functions Using an Evolutionary Intermediate and Multidirectional Search

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