Substrate specificities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenase enzyme systems.
Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (alpha and beta). To assess the contributions of the alpha and beta subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different beta subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.
Parales
R E
RE
Department of Microbiology and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City 52242, USA. rebecca-parales@uiowa.edu
Emig
M D
MD
Lynch
N A
NA
Gibson
D T
DT
D019117:Burkholderia / Q000201:enzymology; D004136:Dinitrobenzenes / Q000378:metabolism; D049308:Dioxygenases; D004926:Escherichia coli / Q000235:genetics; D007211:Indoles / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009281:Naphthalenes / Q000378:metabolism; D009284:Naphthols / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D014050:Toluene / Q000031:analogs & derivatives
D004136:Dinitrobenzenes; D007211:Indoles; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D009281:Naphthalenes; D009284:Naphthols; D011994:Recombinant Proteins; C031721:naphthalene; D014050:Toluene; C016403:2,4-dinitrotoluene; C029955:2-nitrotoluene; C042334:1,2-dihydroxy-1,2-dihydronaphthalene; C030374:indole; D010105:Oxygenases; D049308:Dioxygenases; C100896:2,4-dinitrotoluene dioxygenase; C102756:2-nitrotoluene dioxygenase; C038703:naphthalene dioxygenase; C023172:toluene dioxygenase