Biochemistry: Structural & functional genomics, cofactor and natural product biosynthesis, mechanistic enzymology
Our laboratory utilizes a combination of bioinformatic, biochemical, and biophysical approaches to identify and characterize novel biosynthetic pathways, secondary metabolites, and biocatalysts. The systems under study in our laboratory are chosen for both their biological importance and their potential for employing unusual and interesting enzyme chemistry. Below is an overview of several research projects in our laboratory that are focused on tetrapyrrole biosynthesis. Tetrapyrroles, the ‘pigments of life’, are an important class of biomolecules that function as coenzymes and prosthetic groups. As such, they play essential roles in several fundamental biological processes, such as oxygen and electron transport (heme), photosynthesis (chlorophyll and bacteriochlorophyll), methanogenesis (coenzyme F430), nitrite (heme d1) and sulfite (siroheme) reduction, and fatty and nucleic acid metabolism (cobalamin).
Coenzyme F430 is the nickel corphinoid prosthetic group of methyl-coenzyme M reductase (MCR). MCR is the key enzyme in the biological formation of methane (methanogensis), a potent greenhouse gas and biofuel. MCR has also been shown to catalyze the anaerobic oxidation of methane. In addition to housing coenzyme F430, the active site of MCR contains several unprecedented post-translational modifications, including 2-(S)-methylglutamine, 5-(S)-methylarginine, 3-methylhistidine, S-methylcysteine, and thioglycine residues. The exact roles these post-translational modifications play in MCR catalysis are unknown, as are the identities of the genes responsible for the biosynthesis of the unusual nickel-containing tetrapyrrole. The identification and characterization of the genes responsible for coenzyme F430 biosynthesis and the maturation of MCR may open new avenues in metabolic pathway engineering for the development of designer strains of methanogenic microorganisms for biofuel production. Moreover, detailed studies of the enzymes involved in these processes may aid in the development of specific inhibitors to help reduce natural green house gas emissions and shed light on the novel mechanisms of proteome diversification found in methanogenic archaea.
Heme d1 is the unique cofactor of dissimilatory nitrite reductase (NIR, cytochrome cd1). The reduction of nitrite to nitric oxide by NIR is a key step in the denitrification pathway and an integral component of the global nitrogen cycle. A functional NIR is also important for the virulence of Pseudomonas aeruginosa, an opportunistic human pathogen. Thus, mechanistic studies of the heme d1 biosynthetic enzymes may lead to the development of new antibiotics for the treatment of infections in immunocompromised patients. The genes responsible for heme d1 biosynthesis in P. aeruginosa have been identified and several were found to share sequence homology with genes from a recently discovered heme b biosynthetic pathway. The genes for this alternative heme b pathway are widely distributed in nature, being present in the sequenced genomes of more than a dozen bacterial and archaeal phyla. This suggests that this is an ancient pathway that predates the divergence of the Bacteria and Archaea, and that the distinct functional groups of heme b and heme d1 may arise from divergently evolved enzyme chemistry. In addition, elucidation of the mechanistic details of the alternative heme b biosynthetic pathway, which has yet to be fully characterized and is absent in humans, may lead to the identification of novel drug targets.
Dinoflagellates are an important group of eukaryotic microorganisms found in freshwater and marine environments. Certain taxa produce potent toxins, such as brevetoxin and saxitoxin, which are the causative agents of neurotoxic and paralytic shellfish poisoning. Dinoflagellates are also responsible for red tides, the harmful algal blooms that have a significant negative impact on coastal ecosystems and the health of humans and marine wildlife. Several species of dinoflagellates are both photosynthetic and bioluminescent. The choice between photosynthesis and luminescence is regulated by a cellular circadian biological clock, in which the phase is set by the light-dark cycles of day and night. A key event in the circadian regulation of dinoflagellates is the degradation of chlorophyll to the open-chain tetrapyrrole dinoflagellate luciferin, which is the light emitting molecule utilized by the dinoflagellate luciferase enzyme. The genes and corresponding enzymes responsible for the breakdown of chlorophyll to dinoflagellate luciferin are unknown, though the chemistry involved is likely to resemble that of chlorophyll catabolism in the senescent leaves and ripening fruits of higher plants. Inhibition of this later process results in the accumulation of chlorophyll catabolites that lead to light-induced cell death. Thus, the identification and characterization of the enzymes involved in dinoflagellate luciferin biosynthesis may lead to the development of specific pathway inhibitors, which may find application in the remediation of coastal seawaters. Also, these studies may help facilitate the use of dinoflagellate luciferase as a reporter enzyme and cellular imaging agent.
Chang, W. C., Dey, M., Liu, P., Mansoorabadi, S. O., Moon, S. J., Zhao, Z. K., Drennan, C. L., Liu, H. W. (2013) Mechanistic studies of an unprecedented enzyme-catalysed 1,2-phosphono-migration reaction, Nature 496, 114-118.
Kim, H. J., McCarty, R. M., Ogasawara, Y., Liu, Y. N., Mansoorabadi, S. O., Levieux, J., Liu, H. W. (2013) GenK-catalyzed C-6' methylation in the biosynthesis of gentamicin: isolation and characterization of a cobalamin-dependent radical SAM enzyme, J. Am. Chem. Soc. 135, 8093-8096.
Chang, W. C., Mansoorabadi, S. O., Liu, H. W. (2013) Reaction of HppE with substrate analogues: evidence for carbon-phosphorus bond cleavage by a carbocation rearrangement, J. Am. Chem. Soc. 135, 8153-8156.
Calveras, J., Thibodeaux, C. J., Mansoorabadi, S. O., Liu, H. W. (2012) Sterochemical studies of the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase implicate the FMN coenzyme in substrate protonation, Chembiochem 13, 42-46.
Tang, K. H., Mansoorabadi, S. O., Reed, G. H., and Frey, P. A. (2009) Radical triplets and suicide inhibition in reactions of 4-thia-D- and 4-thia-L-lysine with lysine 5,6-aminomutase, Biochemistry 48, 8151-8160.
Munos, J. W., Pu, X., Mansoorabadi, S. O., Kim, H. J., and Liu, H. W. (2009) A secondary kinetic isotope effect study of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase-catalyzed reaction: evidence for a retroaldol-aldol rearrangement, J. Am. Chem. Soc. 131, 2048-2049.
Mansoorabadi, S. O., Magnusson, O. Th., Poyner, R. R., Frey, P. A. and Reed G. H. (2006) Analysis of the cob(II)alamin – 5'-deoxy-3',4'-anhydroadenosyl radical triplet spin system in the active site of diol dehydrase,Biochemistry 45, 14362-14370.
Behshad, E., Ruzicka, F. J., Mansoorabadi, S. O., Chen, D., Reed, G. H. and Frey, P. A. (2006) Enantiomeric free radicals and enzymatic control of stereochemistry in a radical mechanism: The case of lysine 2,3-aminomutases, Biochemistry 45, 12639-12646.
Mansoorabadi, S. O., Seravalli, J., Furdui, C., Krymov, V., Gerfen, G. J., Begley, T. P., Melnick, J., Ragsdale, S. W. and Reed G. H. (2006) EPR spectroscopic and computational characterization of the hydroxyethylidene-thiamine pyrophosphate radical intermediate of pyruvate:ferredoxin oxidoreductase, Biochemistry 45, 7122-7131.
Sims, P. A., Menefee, A. L., Larsen, T. M., Mansoorabadi, S. O., and Reed, G. H. (2006) Structure and catalytic properties of an engineered heterodimer of enolase composed of one active and one inactive subunit, J. Mol. Biol. 355, 422-431.