Fructans are an important class of plant carbohydrates that consist of linear or
branched chains of fructosyl moieties. Their synthesis requires fructosyltransferases
(FTs) that catalyze the transfer of fructosyl units from a donor substrate (sucrose or
fructan) to an acceptor substrate (sucrose or fructan). The fructosyltransferases involved
in fructan metabolism are related to acid invertases, enzymes that cleave sucrose into
glucose and fructose. An invertase can be considered a fructosyltransferase which
transfers the fructose moiety to water. The aim of the present work was to elucidate what
determines the different catalytic activities of this enzyme group, by use of molecular
methods. In order to study such structure-function relationships we artificially introduced
mutational changes and constructed chimeric FTs (enzymes with exchanged regions).
The goal was to detect the determining regions or single amino acids. For this purpose we
optimized the expression of FTs in the methylotrophic yeast Pichia pastoris and
developed the methodology to create the chimeric constructs. Conventional cloning using
conveniently located restriction sites and the method of overlapping PCR was used.
In a first part domain exchanges between two closely related FTs from cereals
were analyzed by expressing the corresponding constructs in Pichia (Chapter 2). The two
subunits of FTs (N-terminal large subunit and C-terminal small subunit) were exchanged
between Festuca arundinacea (re-classified as Schedonorus arundinaceus)
sucrose:sucrose 1-fructosyltransferases (1-SST) and Hordeum vulgare sucrose:sucrose 6-
fructosyltransferase (6-SFT). The study revealed that it is the large subunit that carries the
structural features responsible for enzyme specificity.
In a second part we focused on the conserved motifs (S/N)DPNG, RDP and EC,
located on the large subunit, that are presumably essential in the active site of plant FTs.
For this purpose two other SST-SFT-chimeras with exchanged N-termini encompassing
these motifs, as well as Festuca 1-SST carrying single amino acid substitutions in the
RDP- and EC-motif were analyzed (Chapter 3). This study revealed the importance of the
three hypothesized active site motifs for the transfructosylation reaction. All three of
them were shown to be important for enzyme activity and/or for specificity.
In a third part, we addressed the question what structural components determine
the relative transferase and hydrolase activities of FTs and vacuolar invertases via a
targeted mutational analysis based on sequence comparisons between vacuolar invertases
and 1-SSTs, the latter an example of a sucrose-using FT (Chapter 4). We chose Allium
cepa invertase and Festuca arundinacea 1-SST for our analysis. Nine amino acids
dispersed along the sequence could be identified correlating with either invertase or
1-SST activities. The selected amino acids of onion invertase were mutated to the
corresponding amino acids in Festuca 1-SST and vice versa. For both enzymes, the
mutations were analyzed independently. Functional expression in Pichia revealed shifts
in the catalytic specificity and activity, demonstrating the importance of these amino
acids outside the three highly conserved motifs (S/N)DPNG, RDP, and EC for the
enzymatic reaction (Chapter 4).
This work helped to narrow down the region potentially responsible for enzyme
specificity in plant FTs. We could pinpoint the importance of the regions with the highly
conserved motifs, and of some additional characteristic single amino acids dispersed
along the sequence, for enzyme activity and specificity
