NICHOL, Tim (2011). Mutagenic studies into the catalytic versatility of soluble methane monooxygenase. Doctoral, Sheffield Hallam University.
Documents
16489:213424
PDF (Version of Record)
30994_Nichol.pdf - Accepted Version
Available under License All rights reserved.
30994_Nichol.pdf - Accepted Version
Available under License All rights reserved.
Download (5MB) | Preview
Abstract
Soluble methane monooxygenase (sMMO) is a multicomponent bacterial enzyme
that catalyzes the oxidation of methane to methanol, as well as oxidizing many other
adventitious substrates. A number of mutagenic studies were carried out on the
sMMO enzyme of Methylosinus trichosporium OB3b in order to gain insight into
sMMO and probe how structural aspects relate to function of the enzyme.
Leu110 within the hydroxylase a-subunit of sMMO has been proposed as a
possible gating residue, controlling access of substrate to the active site
(Rosenzweig et al. 1997). A range of site directed mutants were created at the 110
position and screened for activity with a number of aromatic substrates. All mutants
showed relaxed regioselectivity with all substrates assayed. However no evidence of
a gating residue was found, indicating that Leu110 is more important in determining
regioselectivity than substrate access to the active site.
Comparison to the highly similar butane monooxygenase led to the creation of
three site directed mutants: M184V F282L and C151T. M184V and C151T showed
small changes in regioselectivity and reduced activity with most substrates. The
M184V mutant showed relaxed regioselectivity and a novel oxidation product with
the substrate mesitylene which may have implications for substrate trafficking. The
F282L mutant produced a stable enzyme which had no activity with any of the
substrates tested, showing Phe282 is important for the enzyme function.
A random mutagenesis experiment was devised and a colorimetric screen for
the oxidation of triaromatic compounds was used to screen mutant libraries for
activity towards anthracene and phenanthrene. However no activity towards
triaromatic compounds was detected. In order to improve the cloning strategies and
to make creation of mutant libraries easier, a novel expression vector pT2ML was
created. The pT2ML vector reduces the number of cloning steps required to make
soluble methane monooxygenase mutants. This expression system was used to
make a site directed mutants F188Aand N116G in order to complement previous
site directed mutant studies, as well as a recombinant wild type mutant in order to
asses the activity of the new expression system which is comparible to the wild type
enzyme.
More Information
Statistics
Downloads
Downloads per month over past year
Share
Actions (login required)
![]() |
View Item |