RUI: Characterization and Purification of Mg-protoporphyrin IX Monomethyl Ester Cyclase from Photosynthetic Prokaryotes

Illinois Wesleyan University, Bloomington IL

Investigators

Abstract

The biochemical steps that are required for chlorophyll and bacteriochlorophyll synthesis have been identified and in most cases the genes that encode the various enzymes have been identified. One enzyme that is not as well understood is the Mg-protoporphyrin IX monomethyl ester cyclase. This enzyme converts Mg-protoporphyrin IX monomethyl ester into protochlorophyllide. To further understand this enzyme in plants and bacteria, the following research will be carried out. a) Demonstration of in vitro activity of the anaerobic cyclase from Rhodobacter capsulatus and purification of the enzyme components. Purification of the enzyme will allow determination of cofactor requirements. Determination of the cofactors is key to testing the hypothesis that the anaerobic cyclase utilizes a radical mechanism mediated by adenosylcobalamin. b) Determine components of the anaerobic cyclase by genetic analysis. It is clear from experiments in plant systems that two proteinaceous components are required for cyclase activity. Recent results suggest that two components are required for the anaerobic cyclase activity. Genetic approaches will be used to identify additional loci affecting enzymatic activity. c) Test if the putative chlE gene product from Synechocystis sp. PCC 6803 is a functional homolog to bchE from R. capsulatus. This will be accomplished by expression of the chlE gene in R. capsulatus. This hypothesis will be tested more fully by purifying the ChlE polypeptide and characterizing its function in the cyclase enzyme from Synechocystis sp. PCC 6803. In addition, experiments will be performed to test whether the cyanobacterial enzyme can function anaerobically. d)Involve undergraduate science students in a research project that will prepare them for advanced studies or for teaching at the secondary education level. Chlorophyll is a very important compound because it is used to absorb light for photosynthesis. The goal of this project is to better understand one of the enzymes in chlorophyll synthesis. The enzyme under study is not well understood, but is required for the synthesis of chlorophyll by cells. The enzyme is also limited in iron-poor cells leading to yellowing of the plants. The project will examine the proteins from bacteria that are involved in the reaction. Other components of the cell that are required for this reaction to occur will be identified. BENNING