Download Master project glorin

Master thesis project:
Biosynthesis of the signaling molecule glorin from social amoebae
The social amoebae are a group of unicellular organisms that transiently achieve
multicellularity by aggregation of single cells, aimed at the formation of fruiting bodies in
which dormant spores survive unfavorable conditions. We have previously shown that the
earliest diverged species of social amoebae use the modified dipeptide glorin as a
chemoattractant and signaling molecule that alters gene expression in aggregating cells. A
hallmark of the glorin-based communication system is that the signaling molecule glorin
needs to be degraded extracellularly to allow the amoebae to position in a gradient and move
to aggregation centers. A major goal of a JSMC-funded Ph.D. project is to identify the glorindegrading enzyme from the amoeba Polysphondylium pallidum. In this context we wondered
how glorin might be synthesized. Glorin is an unusual dipeptide in which the γ-carboxyl group
of glutamate is attached to ornithine-1,5-lactam (see figure). Because the P. pallidum
genome does not contain non-ribosomal peptide synthases that might be capable of forming
the amide bond in γ-glutamyl-ornithine-δ-lactam, we want to evaluate whether either of the
five γ-glutamyltranspeptidases (GGTs) enocded in the P. pallidum genome is capable of
forming this unusual dipeptide as a first step of glorin biosynthesis. In the master thesis
project we will first verify the gene annotation of the five putative GGT candidate genes in the
P. pallidum genome and then clone their coding regions into bacterial expressing vectors to
obtain purifed recombinant proteins. An HPLC-UV-based biochemical assay must be
established in which we can test whether cloned proteins are capable of catalyzing the
transfer of the γ-glutamyl moiety of glutathione to either ornithine or ornithine-1,5-lactam.