Expression and substrate specificity of the toluene dioxygenase of Pseudomonas putida NCIMB 11767

1996 ◽  
Vol 45 (1-2) ◽  
pp. 56-62 ◽  
Author(s):  
S. C. Heald ◽  
R. O. Jenkins
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Toluene Dioxygenase

scholarly journals Subtle Difference between Benzene and Toluene Dioxygenases of Pseudomonas putida

2005 ◽  
Vol 71 (3) ◽  
pp. 1570-1580 ◽  
Author(s):  
Claire Bagnéris ◽  
Richard Cammack ◽  
Jeremy R. Mason
Keyword(s):  
Amino Acids ◽  
Electron Transfer ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Amino Acid Sequences ◽  
Subunit Structure ◽  
Subtle Difference ◽  
Α Subunit ◽  
Toluene Dioxygenase

ABSTRACT Benzene dioxygenase and toluene dioxygenase from Pseudomonas putida have similar catalytic properties, structures, and gene organizations, but they differ in substrate specificity, with toluene dioxygenase having higher activity toward alkylbenzenes. The catalytic iron-sulfur proteins of these enzymes consist of two dissimilar subunits, α and β; the α subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity. The amino acid sequences of the α subunits of benzene and toluene dioxygenases differ at only 33 of 450 amino acids. Chimeric proteins and mutants of the benzene dioxygenase α subunit were constructed to determine which of these residues were primarily responsible for the change in specificity. The protein containing toluene dioxygenase C-terminal region residues 281 to 363 showed greater substrate preference for alkyl benzenes. In addition, we identified four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhanced the preference for ethylbenzene. The positions of these amino acids in the α subunit structure were modeled by comparison with the crystal structure of naphthalene dioxygenase. They were not in the substrate-binding pocket but were adjacent to residues that lined the channel through which substrates were predicted to enter the active site. However, the quadruple mutant also showed a high uncoupled rate of electron transfer without product formation. Finally, the modified proteins showed altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains. We propose that these properties can be explained by a more facile diffusion of the substrate in and out of the substrate cavity.

scholarly journals Characterization of Hybrid Toluate and Benzoate Dioxygenases

2003 ◽  
Vol 185 (18) ◽  
pp. 5333-5341 ◽  
Author(s):  
Yong Ge ◽  
Lindsay D. Eltis
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Acinetobacter Calcoaceticus ◽  
The Other ◽  
Β Subunit ◽  
Structural Determinants ◽  
Α Subunit ◽  
Benzoate Dioxygenase

ABSTRACT Toluate dioxygenase of Pseudomonas putida mt-2 (TADOmt2) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADP1) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the α subunit of one enzyme and the β subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADOADP1 utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADOmt2 (3-methylbenzoate > benzoate ∼ 3-chlorobenzoate > 4-methylbenzoate ∼ 4-chlorobenzoate ≫ 2-methylbenzoate ∼ 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the αBβT hybrid oxygenase for these benzoates corresponded to that of BADOADP1, the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the αTβB hybrid oxygenase differed slightly from that of TADOmt2 (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the αTβB hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADOmt2 parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O2 utilization in the hybrid than in the parent. Overall, these results support the notion that the α subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function.

Alteration of the Substrate Specificity of Benzoylformate Decarboxylase from Pseudomonas putida by Directed Evolution

ChemBioChem ◽  
2003 ◽  
Vol 4 (8) ◽  
pp. 721-726 ◽  
Author(s):  
Bettina Lingen ◽  
Doris Kolter-Jung ◽  
Pascal Dünkelmann ◽  
Ralf Feldmann ◽  
Joachim Grötzinger ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Directed Evolution ◽  
Benzoylformate Decarboxylase

Benzylic monooxygenation catalyzed by toluene dioxygenase from Pseudomonas putida

Biochemistry ◽  
1988 ◽  
Vol 27 (4) ◽  
pp. 1360-1367 ◽  
Author(s):  
Lawrence P. Wackett ◽  
Lawrence D. Kwart ◽  
David T. Gibson
Keyword(s):  
Pseudomonas Putida ◽  
Toluene Dioxygenase

scholarly journals Poly-3-hydroxyalkanoate synthases from Pseudomonas putida U: substrate specificity and ultrastructural studies

2007 ◽  
Vol 1 (2) ◽  
pp. 170-176 ◽  
Author(s):  
Sagrario Arias ◽  
Angel Sandoval ◽  
Mario Arcos ◽  
Librada M. Cañedo ◽  
Beatriz Maestro ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Ultrastructural Studies

scholarly journals Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

2012 ◽  
Vol 78 (7) ◽  
pp. 2200-2212 ◽  
Author(s):  
Hannes Leisch ◽  
Rong Shi ◽  
Stephan Grosse ◽  
Krista Morley ◽  
Hélène Bergeron ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Coenzyme A ◽  
Free Acid ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Content Type ◽  
Crystal Forms

ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

scholarly journals Altering the Substrate Specificity of Polyhydroxyalkanoate Synthase 1 Derived from Pseudomonas putida GPo1 by Localized Semirandom Mutagenesis

2004 ◽  
Vol 186 (13) ◽  
pp. 4177-4184 ◽  
Author(s):  
Der-Shyan Sheu ◽  
Chia-Yin Lee
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Chain Length ◽  
Ralstonia Eutropha ◽  
Pha Synthase ◽  
Multiple Point ◽  
Evolution System ◽  
Degenerate Primers ◽  
Short Chain Length ◽  
Single Polymer Chain

ABSTRACT The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 Pp , class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA− as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB−4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB−4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB−4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum.

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