Identifying a new plant metabolic pathway
Our lab focuses on polyamine synthesis and transport so that we can learn how cells sense and respond to these biomolecules.
It had been assumed that there was only a single putrescine synthesis pathway in Arabidopsis, starting with arginine decarboxylase which converts the amino acid arginine to agmaatine and using two additional enzymes to convert agmatine to putrescine
but I wondered why two arginine decarboxylases were needed to funnel substrates into a single pathway
Were there other enzymes capable of converting agmatine to putrescine?
Plant arginases which convert arginine to ornithine are phylogenetically related to bacterial agmatinases : enzymes that convert agmatine to putrescine. We made a 3D model of one of the Arabidopsis arginases using a program called Phyre2 and noted that structurally it retained all the key features of an agmatines.
So it looks very much like an agmatinase, but would it pass the Duck test?
We devised a yeast complementation assay to show when both arginine decarboxylate and an arginase from Arabidopsis were expressed in a yeast mutant, the mutant, is able to synthesis putrescine and grow.
In in vitro assays, we showed that the Km of Arginase enzymes for agmatine is about 70 uM and thus comparable to expected levels for this substrate in plant leaves. By comparison the Km of this enzyme for arginine is about 30 mM.
In Arabidopsis, one arginine decarboxylase and one agmatinase is localized in the chloroplast , so both enzymes needed for putrescine synthesis are in the same organelle.
Relative to many other enzymes, plant arginases are very conserved. We hypothesize that this is a requirement for their dual metabolic roles as both arginases during plant germination and agmatinase enzymes at other times.
This project, the discovery of new pathway in the primary metabolism of the model plant was a team effort of my four Phd graduate students, Jigar Patel Menaka Ariyaratne. Sheaza Ahmed and Lingxiao Ge
