The Bacteroides fragilis NAD-specific Glutamate Dehydrogenase Enzyme is Cell Surface-Associated and Regulated by Peptides at the Protein Level |
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| Institution: | 1. Sustainable Chemistry and Material Resources, Institute of Sustainable and Environmental Chemistry, Leuphana University of Lüneburg, Universitätsallee 1 C13, DE-21335, Lüneburg, Germany;2. Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia, 41522, Egypt;1. Department of Biology, American University of Beirut, Beirut 1107 2020, Lebanon;2. Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan;3. H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan;4. Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21412, Saudi Arabia;1. Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, United States;2. School of Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, United States;3. Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States;4. Doctor’s Data, 3755 Illinois Avenue, St. Charles, IL, United States;5. Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, United States |
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| Abstract: | Bacteroides fragilis has two enzymes with glutamate dehydrogenase (GDH) activity, namely, a dual cofactor NAD(P)H-dependent GDHA, and an NADH-specific GDHB. The presence of two enzymes with the same function is unusual and may play a role in the ability of this organism to survive a variety of environmental conditions. Here we report on the purification and characterisation of the GDHB protein expressed in Escherichia coli from the recombinant plasmid, pGDH15-1 carrying the gdh B gene. The recombinant protein was purified to electrophoretic homogeneity and had a subunit molecular mass of approximately 48 kDa. The temperature and pH activity optima were 38°C and 8.0, respectively, and GDHB enzyme activity was inhibited two-fold by the presence of divalent cations (Ca2+, Mg2+). The presence of monovalent cations (Na+, K+) or metabolites (ATP, AMP, ADP or GTP) did not affect enzyme activity. The regulation of GDHB activity was examined at the protein level and evidence of post-translational regulation of the protein in response to peptides but not ammonia was found. Localisation studies using cell fractions of B. fragilis grown under high peptide conditions showed that 79% of GDHB activity was expressed in the membrane fraction. This result was confirmed by immunogold labelling and electron microscopy of B. fragilis cells. It is possible that the GDHB enzyme might play an important role in bacterial survival during invasion of host tissue through its cell-surface location and its regulation via peptides produced by proteases. |
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