Presentation Title

Purification and Crystallography of Ribofuranosylaminobenzene 5’phosphate (RFAP) Synthase for Future Studies of Inhibition of Biological Methane Production

Faculty Mentor

Madeline Rasche

Start Date

23-11-2019 8:00 AM

End Date

23-11-2019 8:45 AM

Location

51

Session

poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Methane-producing microorganisms (methanogens) have been linked to obesity in humans, which makes it beneficial that growth of these microbes should be inhibited. Methanogens reside in human intestines and rumen of livestock, which are anaerobic environments. Recently, it has been found that methanogens are potential regulators of some metabolic processes and functions associated with digestion. These studies have found that methanogen levels are more elevated in obesity patients and that through antibiotic treatments, the disruption of the bacterial syntrophs might potentially correspond to a lower level of unhealthy cholesterol (low-density lipoproteins, LDL) in the body. Methanogens require a cofactor called tetrahydromethanopterin (H4MPT), which is a one-carbon carrier needed for enzymes involved in methanogenesis. Ribofuranosylaminobenzene 5’phosphate (RFAP) synthase is the first committed enzyme in the nine-step pathway of H4MPT side chain synthesis. The enzyme was acquired through the methanogenic archaeon Methanocaldoccus jannaschii (MJ1427). The purpose of this research is to purify RFAP synthase to homogeneity using affinity and gel filtration chromatography to then determine the three-dimensional structure of potential drug-binding sites using X-ray crystallography. Setting crystal drops of purified RFAP synthase produced rod-like crystals for further analysis. Optimization using a crystallography solution containing lithium sulfate produced a crystal of 3.70-Angstrom resolution. Further optimization and production of heavy atom derivative are underway to solve the structure of RFAP synthase. Analysis of the three-dimensional structure of the active site will assist in the design of potential methanogen inhibitors in the future.

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Nov 23rd, 8:00 AM Nov 23rd, 8:45 AM

Purification and Crystallography of Ribofuranosylaminobenzene 5’phosphate (RFAP) Synthase for Future Studies of Inhibition of Biological Methane Production

51

Methane-producing microorganisms (methanogens) have been linked to obesity in humans, which makes it beneficial that growth of these microbes should be inhibited. Methanogens reside in human intestines and rumen of livestock, which are anaerobic environments. Recently, it has been found that methanogens are potential regulators of some metabolic processes and functions associated with digestion. These studies have found that methanogen levels are more elevated in obesity patients and that through antibiotic treatments, the disruption of the bacterial syntrophs might potentially correspond to a lower level of unhealthy cholesterol (low-density lipoproteins, LDL) in the body. Methanogens require a cofactor called tetrahydromethanopterin (H4MPT), which is a one-carbon carrier needed for enzymes involved in methanogenesis. Ribofuranosylaminobenzene 5’phosphate (RFAP) synthase is the first committed enzyme in the nine-step pathway of H4MPT side chain synthesis. The enzyme was acquired through the methanogenic archaeon Methanocaldoccus jannaschii (MJ1427). The purpose of this research is to purify RFAP synthase to homogeneity using affinity and gel filtration chromatography to then determine the three-dimensional structure of potential drug-binding sites using X-ray crystallography. Setting crystal drops of purified RFAP synthase produced rod-like crystals for further analysis. Optimization using a crystallography solution containing lithium sulfate produced a crystal of 3.70-Angstrom resolution. Further optimization and production of heavy atom derivative are underway to solve the structure of RFAP synthase. Analysis of the three-dimensional structure of the active site will assist in the design of potential methanogen inhibitors in the future.