Benzylsuccinate Synthase (BSS) is a glycyl radical enzyme that catalyzes the fumarate addition reaction which enables anaerobic bio-degradation of hydrocarbons in bacterial cultures. The energetically challenging nature of the fumarate addition reaction and the fact that it is catalyzed by a free radical mechanism, has intrigued researchers in recent times. However, the extreme sensitivity of the glycyl radical to oxygen has precluded structural characterization and detailed experimental investigation of BSS at the molecular level. Here, we present a systematic and comprehensive computational approach involving homology modeling, docking, GPU enabled molecular dynamics (MD) simulations, molecular mechanical generalized Born surface area (MMGBSA) calculations and umbrella sampling techniques to unravel the molecular basis for the fumarate addition reaction in BSS. The active site topology of BSS was elucidated using homology modeling and docking techniques while the overall stability of the free enzyme and the dynamics of substrates (toluene and fumaric acid) bound at the active site was analyzed using MD simulations, performed on GPUs using the cuda version of pmemd in AMBER. The results demonstrate the experimentally observed syn addition of toluene to fumaric acid as well as pin-point specific protein-substrate interactions that stabilize the substrates at the active site. The umbrella sampling simulations also reveal that substrate binding favorably impacts the active site dynamics so as to enable feasible radical transfer pathways for the proposed fumarate addition reaction mechanism.

Unraveling the fumarate addition reaction in the glycyl radical enzyme Benzylsuccinate Synthase: A GPU enabled comprehensive computational study

248th National Meeting (2014)

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