Characterisation of class II and III fructose bisphosphatases in Clostridium acetobutylicum ATCC 824.

Almohaish, Ali Abdullah (2012) Characterisation of class II and III fructose bisphosphatases in Clostridium acetobutylicum ATCC 824. PhD thesis, Edinburgh Napier University.

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In low GC Gram-positive bacteria, the phenomenon of carbon catabolite repression is dependent on a metabolite-activated bifunctional protein kinase/phosphorylase. In Clostridium acetobutylicum ATCC 824, the hprK gene encodes the bifunctional protein kinase/phosphorylase that is dependent on fructose 1,6-bisphosphate for activity. However, a putative glpX class II gene that might encode for fructose 1,6-bisphosphatase is located upstream of hprK and to date this is a unique gene arrangement. Therefore, the product of the putative glpX gene might have a vital role in regulating the activity of bifunctional protein kinase/phosphorylase by hydrolysing fructose 1,6-bisphosphate which is needed for the kinase activity. In the present work, experimental evidence is presented that the putative glpX gene encodes a fructose 1,6-bisphosphatase which can hydrolyse fructose 1,6-bisphosphate to fructose 6-phosphate and phosphate. FBPase activity was first demonstrated by cloning and transforming the glpX gene into an E. coli fbp mutant which cannot grow on gluconeogenic substrates such as glycerol. The transformed glpX gene was able to complement the fbp mutation of E. coli for growth on glycerol. GlpX was overexpressed as a GST-fusion protein and purified, and activity was demonstrated using fructose 1,6-bisphosphate as substrate. Activity was assayed at pH 8.0, and in the presence of Mn2+, but the enzyme was inhibited completely by 1 mM phosphate. A putative fbp class III gene was also overexpressed and the encoded protein was purified as a GST-fusion product in order to compare Fbp with GlpX. To our knowledge, this is the first study that was able to purify a Fbp class III enzyme. The Fbp protein showed almost the same behaviour towards inhibitors compared to GlpX, but had a considerably higher specific activity than GlpX under the conditions of the experiments. The glpX gene was shown by RT-PCR to be transcribed together with hprK on the same mRNA during growth on glucose, and this indicates that glpX is unlikely to be a gluconeogenic gene. The results suggest that GlpX may play a novel and specific role in regulating the kinase activity activity of bifunctional protein kinase/phosphorylase and carbon catabolite repression in C. acetobutylicum.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Clostridium acetobutylicum ATCC 824; gene coding; fructose; kinase activity; carbon catabolite repression;
University Divisions/Research Centres: Faculty of Health, Life & Social Sciences > School of Life, Sport and Social Sciences
Dewey Decimal Subjects: 500 Science > 570 Life sciences; biology > 572 Biochemistry
Library of Congress Subjects: Q Science > QD Chemistry
Item ID: 5692
Depositing User: Mrs Lyn Gibson
Date Deposited: 22 Oct 2012 13:17
Last Modified: 30 Jun 2014 00:38

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