Methyl-coenzyme M Reductase
Methyl-coenzyme M reductase (MCR) is the enzyme involved in biological methane production and anaerobic methane activation. The reaction that is catalyzed is the reduction of methyl-coenzyme M (CH3-S-CoM) by coenzyme B (HS-CoB) resulting in the formation of CH4 and the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB). Central to the function of MCR is the nickel-containing tetrapyrrole cofactor 430 (F430). Coenzyme F430 is a nickel porphinoid of unique structure. The pi-chromophore extends only over three of the four nitrogens, making F430 the most extensively reduced tetrapyrrole found in nature.
Our research has several goals. First, we want to understand the mechanistic details of the reversible reaction. Secondly, we want to understand the mechanism of activating the enzyme. The enzyme is active when the nickel is in the Ni(I) state. The midpoint potential for the Ni(I)/Ni(II) transition is estimated to be -650 mV while the source of electrons has to come from the H2/H+ couple that under growth conditions has a midpoint potential of -414 mV.
The molecular biological part of this project is done in collaboration with Sang-Jin Suh. Working on this project is Divya Prakash
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This project received funding from the Petroleum Research Fund |
Isoprene Synthesis
In nature, there are two pathways found for the synthesis of the isoprene precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). These two compounds are very important as the building blocks for the essential biological molecules called isoprenoids, which include vitamins, cholesterol, steroid hormones, carotenoids and quinones. Mammals, including humans, use the mevalonate pathway to synthesize the isoprene precursors, while eubacteria and some other microorganisms use the DOXP/MEP pathway as the sole pathway for isoprene synthesis. Several of the microorganisms that utilize the DOXP/MEP pathway are pathogens, causing, for example, malaria, multidrug resistant tuberculosis (MDR-TB), anthrax, plague, gastro-intestinal ulcers and venereal diseases. This makes the DOXP/MEP pathway an attractive target for the development of new anti-infective drugs. Since this pathway is not present in humans these inhibitors should demonstrate very low toxicity.
Detailed knowledge of the mechanism and regulation of the DOXP pathway is a prerequisite for the rational design of inhibitors that are potential candidates for new anti-infective drugs; however, due to its recent discovery the function and catalytic mechanism of some of the proteins in this pathway are not well understood. The goal of the proposed research is to understand the reaction mechanism of the last two proteins in the DOXP pathway, IspG ((E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; also known as GcpE) and IspH ((E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; also known as LytB).
Both enzymes contain an iron-sulfur cluster in their active site which appears to be involved in direct binding of the respective substrates. Although both enzyme catalyze a similar step, the reductive removal of a hydroxyl group, the proposed reaction intermediates are very different. Our research is focused on understanding these differences and to obtain a full understanding of the different reaction steps in both mechanisms.
The synthesis part of the project is done in collaboration with Forrest Smith. Working on this project are Selamawit Ghebreamlak and Xiao Xiao
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This project received funding from NSF. |
Electron Bifurcation
Electron bifurcation has now been shown for several protein complexes and is expected to be a wide spread process in nature. Electron bifurcation enables enzymes to create electrons with a very low redox potential without the need for coupling of the electron transfer step to ATP hydrolysis. The bifurcation process always involves a quinone or flavin group. Of two electrons that enter the bifurcation process at a certain potential, one comes out at a much lower potential while the other comes out at a much higher potential. There is a tight couple in this process: each type of electron will not be produced when the other is not simultaneously generated. The discovery of this process in the hydrogenase:heterodisulfide reductase complex in methanogenic Archaea is the first proof that electron bifurcation plays an important role in anaerobic biological energy transduction.
The overall goal of this project is to get a detailed understanding of the electron bifurcation process in the heterodisulfide reductase complexes from Methanothermobacter marburgensis and Methanococcus maripaludis. The is a collaborative project with the group of John Leigh.
Working on this project is Mohiuddin Ovee. |
RNA Polymerase
This is a project in collaboration with the group of Dan Lessner. The RNA polymerases of Methanosarcina acetivorans contain regulatory iron-sulfur clusters. The clusters are proposed to function by modulating the structure and activity of the protein in response to oxygen or reactive oxygen species. The results obtained will provide insight into the function of iron-sulfur clusters in proteins involved in response to oxidative stress.
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Working on this project is Divya Prakash
This project is funded by NASA. |
Acetyl-CoA Synthase
This is a project in collaboration with the group of David Grahame. Acetyl-CoA synthesis from one-carbon precursors (and also cleavage of acetyl-CoA to yield C-1 products) is catalyzed by a multi-enzyme complex, known as ACDS (the acetyl-CoA decarbonylase/synthase complex). Both the carbon-sulfur and the carbon-carbon bond of acetyl-CoA are formed by the ACDS complex in a highly unusual biochemical mechanism involving metal-based carbonyl group insertion, and/or methyl group migration.
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Working on this project is Selamawit Ghebreamlak |