UNIVERSITY OF MALTA
RESEARCH SEMINARS
Abstract form
Title: SOD FeMn-ism
or
Selectivity and specificity of iron and manganese in the mononuclear
superoxide dismutases of Escherichia coli.
Presenter: Prof. Gary J. Hunter
Contact address: Department of Physiology and Biochemistry, University of Malta
Tel: 23402917
Fax:
Email: gary.hunter@um.edu.mt
Presentation date: 4th April 2005
(approximately 200-250 words)
Abstract
Superoxide dismutases (SODs) are found in one form or another in all organisms that utilise
molecular oxygen, reflecting the fact that it is relatively easy for a molecule of oxygen to
pick up an electron, converting it into the dangerous, reactive radical, superoxide. Three
classes of enzymes with SOD activity have been discovered and can be classified as
dinuclear (copper and zinc -containing), mononuclear (manganese or iron –containing) and
those containing nickel. Despite a very high degree of sequence and structural similarity, the
mononuclear superoxide dismutases exhibit an absolute requirement for their specific metal
ion. Using the iron and manganese SODs of Escherichia coli, we are investigating the
sequence and structural requirements which dictate (i) the selection of metal ion during
protein expression and (ii) the specificity for metal ion during catalysis. These two processes
may not necessarily be the same.
We have cloned the genes for sodA (MnSOD) and sodB (FeSOD) from E.coli into a highly
efficient expression vector developed in our laboratory. Protein purification has been
achieved through metal chelate chromatography, accomplished by the inclusion of a
hexahistidine tag into the expressed protein at its N-terminus. Site-directed mutagenesis has
been used with these genes to produce mutant SOD proteins containing specific amino acid
changes. Selectivity of site-directed mutants for metal ion is being examined by expression
of protein in bacterial cultures grown in minimal media supplemented with iron, manganese
or both metals. Specificity is being examined using enzymological techniques including
zymography and spectrophotometry. Crystallisation of selected mutant enzymes is underway
and X-ray analysis has begun.