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

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

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.

Dinoflagellate Luciferin

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.

Donnan, P. H. and Mansoorabadi, S. O. (2022) Broken-Symmetry Density Functional Theory Analysis of the Ω Intermediate in Radical S-Adenosyl-l-Methionine Enzymes: Evidence
for a Near-Attack Conformer over an Organometallic Species, J. Am. Chem. Soc. 144, 3381-3385.
 
Li, S., Isiorho, E. A., Owens, V. L., Donnan, P. H., Odili, C. L., and Mansoorabadi, S. O. (2021) A noncanonical heme oxygenase specific for the degradation of c-type heme, J. Biol. Chem. 296, 100666.
 
Sahrmann, P. G., Donnan, P. H., Merz, K. M. Jr, Mansoorabadi, S. O., and Goodwin, D. C. (2020) MRP.py: A Parametrizer of Post-Translationally Modified Residues, J. Chem. Inf. Model. 60, 4424-4428.
 
Ghebreamlak, S. M., Mansoorabadi, S. O. (2020) Divergent Members of the Nitrogenase Superfamily: Tetrapyrrole Biosynthesis and Beyond, ChemBioChem 21, 1723-1728.
 
Donnan, P. H., Ngo, P. D., and Mansoorabadi, S. O. (2018) Constant pH Accelerated Molecular Dynamics Investigation of the pH Regulation Mechanism of Dinoflagellate Luciferase, Biochemistry 57, 295-299. (‘Future of Biochemistry’ Special Issue)
 
Ngo, P. D. and Mansoorabadi, S. O. (2017) Investigation of the Dinoflagellate Bioluminescence Mechanism: Chemically Initiated Electron Exchange Luminescence or Twisted Intramolecular Charge Transfer?, ChemPhotoChem 1, 383-387.
 
Mansoorabadi, S. O., Zheng, K., and Ngo, P. D. (2017) Biosynthesis of Coenzyme F430 and the Post-Translational Modification of the Active Site Region of Methyl-Coenzyme M Reductase, in Metalloprotein Active Site Assembly (Johnson, M. K. and Scott, R. A., Eds.), John Wiley & Sons, Ltd, West Sussex, UK.
 
Zheng, K., Ngo, P. D., Owens, V. L., Yang, X. P., and Mansoorabadi, S. O. (2016) The biosynthetic pathway of coenzyme F430 in methanogenic and methanotrophic archaea, Science 354, 339-342.
 
Ngo, P. D., Mansoorabadi, S. O., and Frey, P. A. (2016) Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts, J. Phys. Chem. B, 120, 7353-7359.
 
Chang, W. C., Dey, M., Liu, P., Mansoorabadi, S. O., Moon, S. J., Zhao, Z. K., Drennan, C. L., and Liu, H. W. (2013) Mechanistic studies of an unprecedented enzyme-catalysed 1,2-phosphono-migration reaction, Nature 496, 114-118.