Project 5 - Kinetic characterisation of multiple isoforms of sucrose synthase in plants
Sucrose synthase is the main enzyme of sucrose metabolism in non-photosynthetic cells. By catalysing the conversion of sucrose to UDPglucose and fructose it allows imported carbon to enter the metabolism of these cells. Many plants contain multiple isoforms of sucrose synthase which may have different physiological roles. In collaboration with researchers at the John Innes Institute, we have recently cloned genes encoding three isozymes of the enzyme from pea (Sus 1-3), and shown that these are differentially expressed, both spatially and temporally. Measurement of kinetic constants of the isozymes purified from an E. coli expression system shows that each has distinct properties. Studies on the rug4 mutant of pea indicate that this locus lies within the gene encoding the Sus1. Mutant plants carrying the rug4-c allele lack Sus1 protein and have dramatically lower sucrose synthase activity in the developing embryo and in Rhizobium-infected root nodules, but much smaller effects on activity in other tissues. Intriguingly, a second allele (rug4-a) has very low activity when assayed in the direction of sucrose cleavage, but almost normal activity in the synthetic direction (Barratt et al. (2001) Plant Physiol. 127, 655-664). We are now extending this study by isolating "knockout" mutants in each of the six genes encoding sucrose synthase in Arabidopsis to establish directly the metabolic and physiological roles of each of the isozymes in this model plant.
The aim of this project will be to determine the kinetic properties of the six isozymes of sucrose synthase in Arabidopsis, and relate these to the presumed physiological roles of the different forms based on expression pattern of the genes and the metabolic characteristics of the "knockout" lines. As with our earlier work on pea, the Arabidopsis isozymes will be isolated by PCR, expressed as recombinant proteins in E. coli and purified for kinetic studies. Additionally, we may chose to examine the kinetic properties of the rug4-a isozyme of Sus1 from pea. The molecular details of the point mutation in this protein have been elucidated, but as yet we have no idea of the kinetic basis of the rather curious effects on enzyme activity. Comparison of the kinetic properties of the mutant and wildtype forms of Sus1 would provide valuable information in our attempts to understand the structure-function relationships of this metabolically important enzyme. The work will continue to be conducted in close collaboration with the John Innes Centre, and the project provides an ideal opportunity for a student wishing to pursue a research career in plant science and establish contact with this internationally renowned institute.