Cloning, expression, purification, and characterization of putative cyclooxygenases from bacterial genomes.
Cyclooxygenases are an intensely studied class of enzymes in mammalian systems. They are needed for the synthesis of prostaglandins, local messengers involved in pain, swelling, and blood vessel dilation. However, functional cyclooxygenases have been identified in invertebrates and, more recently, red algae. Open reading frames encoding for proteins predicted to be structurally similar to cyclooxygenases have also been identified.
Our lab is currently subcloning the open reading frames of several of these bacterial proteins into expression vectors and expressing these proteins in large amounts. Protein expression has some associated challenges; the mammalian protein is membrane associated, but the bacterial proteins have altered membrane binding domains that limit their aqueous solubility.
Gupta, K., and Selinsky, B.S. Bacterial and algal orthologs of prostaglandin H2 synthase: novel insights into the evolution of an integral membrane protein. Biochim. Biophys. Acta, 1848, 83-94, 2015.
Gupta, K., Selinsky, B.S., and Loll, P.J. 2.0 A Crystal structure of prostaglandin H2 synthase-1 reconstituted with a manganese porphyrin cofactor. Acta Crystallograpica D, 62, 151-156, 2006.
Gupta, K., Selinsky, B.S., Kaub, C.J., and Loll, P.L. The 2.0 A resolution crystal structure of prostaglandin H2 synthase-1: Structural insights into an unusual peroxidase. J. Mol. Biol., 335, 503-518, 2004.
Gupta, K., Kaub, C.J., Carey, K.N., Casillas, E.G., Selinsky, B.S., and Loll, P.J. Manipulation of kinetic profiles in 2-aryl propionic acid cyclooxygenase inhibitors. Bioorg. Med. Chem. Lett., 14, 667-671, 2004.
Selinsky, B.S., Gupta, K., Sharkey, C.T., and Loll, P.J. Structural analysis of NSAID binding by prostaglandin H2 synthase: Time-dependent and time-independent inhibitors elicit identical enzyme conformations. Biochemistry, 40, 5172-5180, 2001.
Loll, P.J., Sharkey, C.T., O’Connor, S.J., Dooley, C.M., O’Brien, E., Devocelle, M., Nolan, K.B., Selinsky, B.S., and Fitzgerald, D.J. O-Acetylsalicylhydroxamic acid, a novel acetylating inhibitor of prostaglandin H2 synthase: Structural and functional characterization of enzyme-inhibitor interactions. Mol. Pharmacol., 60, 1407-1413, 2001.
Loll, P.J., Kaplan, J., Selinsky, B.S., and Axelsen, P.H. Vancomycin binding to low affinity ligands: Delineating a minimum set of interactions necessary for high affinity binding. J. Med. Chem., 42, 4714-4719, 1999.
Synthesis of aminosterols and characterization of these as membrane lytic agents.
An aminosterol is comprised of a cholestane-like ring system with an attached amine or polyamine substituent. The most well known aminosterol is squalamine, isolated from the dogfish shark. The function of squalamine is still under investigation, but it does possess antimicrobial activity, and may contribute to the immune system of the dogfish shark. We have contributed to the total synthesis of squalamine, and have also synthesized a variety of squalamine analogs for antimicrobial testing.
Our lab is currently synthesizing aminosterols based upon a slightly different cholestane backbone, which may be relevant to mammalian systems. We are also interested in synthesizing aminosterols with attached chromophores for biophysical studies.
Tessema, T-D., Gassler, F., Shu, Y., Jones, S., and Selinsky, B.S. Structure-activity relationships in aminosterol antibiotics: The effect of stereochemistry at the 7-OH group. Bioorganic. Med. Chem. Lett., 23, 3377-3381, 2013.
Xia, J., Chen, Y., Liberatore, K.M., and Selinsky, B.S. The application of diethylaminosulfur trifluoride in the synthesis of fluorinated sterols and bile acids. Tetrahed. Lett., 44, 9295-9297, 2003.
Shu, Y., Jones, S.R., Kinney, W.A., and Selinsky, B.S. The synthesis of spermine analogs of the shark aminosterol squalamine. Steroids, 67, 291-304, 2002.
Selinsky, B.S., Smith, R., Frangiosi, A., Vonbaur, B., and Pedersen, L. Squalamine is not a proton ionophore. Biochim. Biophys. Acta, 1464, 135-141, 2000.
Selinsky, B.S., Zhou, Z., Fotjik, K.G., Jones, S.R., Dollahon, N.R., and Shinnar, A.E. The aminosterol antibiotic squalamine permeabilizes large unilamellar vesicles. Biochim. Biophys. Acta 1370, 218-234, 1998.
Jones, S.R., Selinsky, B.S., Rao, M.N., Zhang, X., Kinney, W.A., and Tham, F.S. Efficient route to 7a-(benzoyloxy)-3-dioxolane cholestan-24(R)-ol, a key intermediate in the synthesis of squalamine. J. Org. Chem., 63, 3786-3789, 1998.
Jones, S.R., Kinney, W.A., Zhang, X., Jones, L.M., and Selinsky, B.S. The synthesis and characterization of analogs of the antimicrobial compound squalamine: 6b-hydroxy-3-aminosterols synthesized from hyodeoxycholic acid. Steroids 61, 565-571, 1996.