Prokaryotic Expression Of GDCT And SHMT From Arabidopsis Thaliana And Site-directed Mutation

Folate is an important coenzyme of two proteins, glycine decarboxylase complex T-protein (GDCT)and serine hydroxymethyltransferase (SHMT), in photorespiration pathway. During the conversionprocess of glycine to serine, these proteins are responsible for transferring methylene group to folates.GDCT protein is responsible for accepting amino methyl group delivered by glycine decarboxylasecomplex, cleaving the amino group, and transferring methylene to folate group; SHMT protein isresponsible for accepting methylene group from folates, and making use of glycine to generate serine.The longer are polyglutamate tails of folates, the stronger is enzymatic activity of these enzymes. Thisstudy focus on the interaction between folates and these two proteins by the method of homologousmodeling, and site-directed mutation of amino acids in GDCT and SHMT proteins that interact withfolate polyglutamate tails. Prokaryotic expression of these two proteins and the estabolishment ofenzymatic activity assay are to verify the interaction between folates and proteins. The results weachieved are listed as below:(1) We cloned GDCT and SHMT genes from leaf cDNA of Arabidopsis thaliana, and constructedGDCT-pET30a(+) and SHMT-pET26b(+) recombinant plasmids;(2) Prokaryotic expressed of GDCT and SHMT proteins the purification of SHMT protien;(3) Enzymatic activity assay of SHMT protein;(4) Homologous modeling of GDCT and SHMT proteins, identify amino acids that interact with folatepolyglutamate tail.This study established the method of performing enzymatic activity assay of SHMT protein. Theinteractions were studied between these two proteins and folates by stimulating three dimensionstructures of SHMT and GDCT protein, identifing amino acids that interact with folate polyglutamatetails and try to mutate these amino acids. This work provides the basic information of potentialmolecular mechanism about how enzymatic activities of SHMT and GDCT proteins rely on folate ascoenzyme during photorespiration pathway.