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Title: Characterisation of a novel non-heme dioxygenase
Author: Wallis, Mary Grace
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1990
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A purification procedure has been developed for a novel extradiol dioxygenase, designated as 3-methylcatechol 2,3-dioxygenase. The enzyme which is expressed in Escherichia coli, was originally derived from a Pseudomonas putida strain able to grow on toluidine. 3-Methylcatechol 2,3-dioxygenase was purified to homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Physical and kinetic properties of the purified enzyme were investigated. The enzyme consists of a single subunit type of Mr = 33,500 ± 2,000 by SDS-PAGE. Gel filtration indicated a molecular weight, under non-denaturing conditions, of 120,000 ± 20,000 consistent with the native enzyme existing as a tetramer of identical subunits. The NH2-terminal sequence (35 residues) has been determined and shows 50% identity with other extradiol dioxygenases. The structural characterisation of 3-methylcatechol 2,3-dioxygenase at the primary, secondary and quaternary levels indicates that the enzyme is typical of the extradiol dioxygenases. The kinetics of 3-methylcatechol 2,3-dioxygenase were investigated using UV/visible spectrophotometry and oxygen electrode polarography. Measurements were made under both standard and modified conditions. Typical saturation kinetics were observed for catechol, 3-methylcatechol and 4-methylcatechol as substrates. Data were analysed to give values of Vmax and Km. The substrate specificity for this enzyme was somewhat different from that seen for other catechol 2,3-dioxygenases, with 3-methylcatechol being cleaved at the highest rate. The Km values for the organic substrates were all around 0.3 μM, the lowest found for any dioxygenase to date. The K_m for dioxygen was determined to be ≤ 10-6M. The enzyme consumed one mole of oxygen per mole of substrate in all three cases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available