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Please use this identifier to cite or link to this item: http://repository.iitr.ac.in/handle/123456789/26954
Title: Retuning rieske-type oxygenases to expand substrate range
Authors: Mohammadi M.
Viger J.-F.
Kumar, Pravindra R.Manish
Barriault D.
Bolin J.T.
Sylvestre M.
Published in: Journal of Biological Chemistry
Abstract: Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE p4, a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE LB400 by the double substitution T335A/F336M, and BphAE RR41, obtained by changing Asn 338, Ile 341, and Leu 409 of BphAE p4 to Gln 338, Val 341, and Phe 409, metabolize dibenzofuran two and three times faster than BphAE LB400, respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE LB400 showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reactioncritical atoms or groups. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
Citation: Journal of Biological Chemistry, 286(31): 27612-27621
URI: https://doi.org/10.1074/jbc.M111.255174
http://repository.iitr.ac.in/handle/123456789/26954
Issue Date: 2011
Keywords: Biphenyl dioxygenase
Burkholderia
Catalytic cavities
Conformational change
Dibenzofurans
Double substitution
Enhanced catalytic activity
Fine Chemicals
Induced-fit mechanism
Oxygenases
Segment lining
Steady-state kinetics
Substrate range
Subunit assembly
Alignment
Catalyst activity
Enzymes
Ionic liquids
Synthesis (chemical)
Substrates
biphenyl dioxygenase
BphAEp4
BphAERR41
dibenzofuran
oxygenase
Rieske type oxygenase
unclassified drug
amino acid substitution
article
Burkholderia
Burkholderia xenovorans
catalysis
conformational transition
controlled study
crystal structure
enzyme activity
enzyme specificity
enzyme stability
enzyme substrate
enzyme substrate complex
oxidation
priority journal
protein assembly
steady state
structure analysis
Burkholderia
Crystallography, X-Ray
Electrophoresis, Polyacrylamide Gel
Gas Chromatography-Mass Spectrometry
Kinetics
Models, Molecular
Oxygenases
Substrate Specificity
Burkholderia xenovorans
ISSN: 219258
Author Scopus IDs: 23390544500
37038864200
55064809000
6602879821
57197844041
7005984229
Author Affiliations: Mohammadi, M., INRS-Institut Armand- Frappier, Laval, QC H7V 1B7, Canada
Viger, J.-F., INRS-Institut Armand- Frappier, Laval, QC H7V 1B7, Canada
Kumar, P., Department of Biological Sciences, Center for Cancer Research, Purdue University, West Lafayette, IN 47907, United States, Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, India
Barriault, D., INRS-Institut Armand- Frappier, Laval, QC H7V 1B7, Canada
Bolin, J.T., Department of Biological Sciences, Center for Cancer Research, Purdue University, West Lafayette, IN 47907, United States
Sylvestre, M., INRS-Institut Armand- Frappier, Laval, QC H7V 1B7, Canada
Funding Details: 
Corresponding Author: Sylvestre, M.; INRS-Institut Armand- Frappier, Laval, QC H7V 1B7, Canada; email: Michel.Sylvestre@iaf.inrs.ca
Appears in Collections:Journal Publications [BT]

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