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Identification and characterization of a novel monooxygenase from Burkholderia xenovorans LB400
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Montes-Matias, Marie Carmen. Identification and characterization of a novel monooxygenase from Burkholderia xenovorans LB400. Retrieved from https://doi.org/doi:10.7282/T36T0MXS

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Uniform TitleIdentification and characterization of a novel monooxygenase from Burkholderia xenovorans LB400
NameMontes-Matias, Marie Carmen (author); Zylstra, Gerben (chair); Chase, Theodore (internal member); Bini, Elisabetta (internal member); Kobayashi, Donald (outside member); Rutgers University; Graduate School - New Brunswick
Date Created2008
Other Date2008-10 (degree)
SubjectMicrobiology and Molecular Genetics, Monooxygenases
Extentxi, 128 pages
DescriptionWhole genome sequences have been key elements in discovering new genes and in predicting protein function. A gene found in Burkholderia xenovorans LB400 encodes a protein that is 77% identical and 85% similar to the p-cymene monooxygenase from Pseudomonas putida F1. Phylogenetic analysis of this enzyme reveals that it is closely related to cymene, xylene and alkane monooxygenase. Protein alignment of these sequences identified 10 conserved histidine residues, of which 8 were reported to be essential for catalysis in alkane monooxygenase. The identified consensus sequence for these enzymes is [HX(3)HX(25)HX(3)HHX(139)HX(2)HH], which is characteristic of membrane-bound hydroxylases with an active site that require iron and the activation of molecular oxygen for their catalytic cycle. In order to analyze the B. xenovorans LB400 monooxygenase enzyme in more detail, we cloned the cognate genes into the expression vector pQE.30 and performed biotransformation assays to determine the substrate specificity of the CymA1A2-like enzyme. The novel monooxygenase found in LB400 hydroxylates p-cymene to the product p-cymen-8-ol. The cymene monooxygenase found in P. putida F1 hydroxylated the methyl group of p-cymene, while the novel monooxygenase catalyzed the hydroxylation of the carbon of the isopropyl group adjacent to the benzene ring. The enzyme also oxidized the substrates cumene, isobutylbenzene, n-butylbenzene, propylbenzene, 1, 4-diisopropylbenzene, tert-butyltoluene, sec-butylbenzene, and 4-ethyltolunene in addition to p-cymene. The collected data indicate that the LB400 monooxygenase described above is able to catalyze an interesting and novel reaction, but its physiological role is still unidentified. Steric hindrance and chain length definitely play an important role in determining the enzyme specificity. Metabolite analysis and RT-PCR data indicated that there is no correlation between the novel enzyme activity and growth of the strain on p-cymene, propylbenzene, 1,4-diisopropylbenzene, 4-ethyltoluene, n-butylbenzene, isobutylbenzene, sec-butylbenzene, tert-butylbenzene, and tert-butyltoluene. Other pathways and enzymes are involved in the degradation of these substrates. This data indicates that although bioinformatic analysis is a valuable tool in protein analysis and function prediction, it cannot always accurately predict the catalytic activity and physiological role of an enzyme.
NotePh.D.
NoteIncludes bibliographical references (p. 120-127).
Genretheses, ETD doctoral
Persistent URLhttps://doi.org/doi:10.7282/T36T0MXS
LanguageEnglish
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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