Computational Studies of Dinoflagellate Luciferase and Radical S-adenosyl-L-methionine Enzymes

Abstract

This dissertation seeks to apply computational methods to study two enzymatic systems: dinoflagellate luciferase and radical S-adenosyl-L-methionine enzymes. Dinoflagellates are microorganisms, many of which are capable of bioluminescence. Their bioluminescence is ‘simple,’ requiring only the luciferase enzyme, the luciferin substrate, and molecular oxygen—no other cofactors or cosubstrates are needed. The enzyme is regulated by pH, active at acidic pH and inactive at alkaline pH. To study its pH-dependent dynamics, constant pH accelerated molecular dynamics was employed. This dissertation reports an open, presumed-active conformer of the enzyme which, to date, experimental structural methods have been unable to obtain. Then, using the open conformer of luciferase, molecular dynamics studies of the reaction substrate, product, and proposed intermediates were conducted. Using the results of these studies, this dissertation pieces together the likely stereochemical and regiochemical course of the dinoflagellate luciferase reaction, predicting which amino acid residues of the enzyme are important for catalysis. Turning to radical S-adenosyl-L-methionine (SAM) enzymes, this dissertation applies broken-symmetry density functional theory to understand the common catalytic steps utilized by members of this diverse enzyme superfamily, which result in the creation of the strong oxidant 5'-dAdo·. This dissertation suggests that a recently discovered intermediate in the common catalytic mechanism, Ω, is not organometallic but is instead a near-attack conformer of SAM bound to the catalytic iron- sulfur cluster. The dissertation applies the same methodology to the case of a non- canonical radical SAM enzyme, Dph2, which does not form 5'-dAdo·. Predicted paramagnetic reaction intermediates are analyzed and compared to experimental data.