UNIVERSITY OF MALTA
LIFE SCIENCE RESEARCH SEMINARS
Web: http://www.um.edu.mt/events/scisem/
Email: scisem@um.edu.mt
Abstract form
Title: Crystallography and physicochemical characterisation of
C. elegans superoxide dismutase mutant proteins
Presenter: Rosalin Bonetta
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Email: rosalin.bonetta@um.edu.mt
Presentation date: 28th April 2014
Abstract
Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme which
catalyses the dismutation of the superoxide radical anion to produce molecular
oxygen and hydrogen peroxide by-products. MnSOD is present in the mitochondria
to protect against the oxidative stress generated during cellular respiration. In
various types of cancer, MnSOD demonstrates tumour suppressor properties. Davis
et al. (2004) revealed that an active site H30N mutant of the human MnSOD has
anti-proliferative effects in vitro as well as anti-tumour effects in vivo.
Kinetic studies revealed a C. elegans MnSOD-3 catalysis mechanism which
resembles the human H30N MnSOD mutant mechanism. For this reason, various C.
elegans H30 mutants were studied to understand the subtle structural differences in
the wild type that have such pronounced effects on catalysis. The C. elegans H30N
mutant is very different from the human H30N mutant, highlighting the fact the wild
type proteins are more different then they appear.
Three H30 active site mutants of C. elegans MnSOD-3 were generated, sequenced
and over-expressed in Escherichia coli. The recombinant proteins were purified
using affinity chromatography and gel filtration. The physicochemical properties of
the wild type MnSODs and the three mutants were characterised. X-ray
crystallography was performed and the structure of C. elegans H30F, H30Q and
H30N MnSOD-3 mutants was solved at 1.6Å resolution. HFEPR studies were
carried out to study the protein active site electron environment. As a biological
assay, the effect of C. elegans MnSODs on the proliferation of chronic myelogenous
leukemia K652 cells was studied. The wildtype isoforms reduced proliferation of
K652 cells in a dose dependent manner.