Exploring the Catalytic Mechanism of Alkanesulfonate Monooxygenase Using Molecular Dynamics

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• 作者：Kira Armacost ; Jonathan Musila ; Symon Gathiaka ; Holly R. Ellis ; Orlando Acevedo
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：May 27, 2014
• 年：2014
• 卷：53
• 期：20
• 页码：3308-3317
• 全文大小：671K
• 年卷期：v.53,no.20(May 27, 2014)
• ISSN：1520-4995

文摘

The complex mechanistic properties of alkanesulfonate monooxygenase (SsuD) provide a particular challenge for identifying catalytically relevant amino acids. In response, a joint computational and experimental study was conducted to further elucidate the SsuD mechanism. Extensive unbiased molecular dynamics (MD) simulations were performed for six SsuD systems: (1) substrate-free, (2) bound with FMNH₂, (3) bound with a C4a-peroxyflavin intermediate (FMNOO^{鈥?/sup>), (4) bound with octanesulfonate (OCS), (5) co-bound with FMNH₂ and OCS, and (6) co-bound with FMNOO鈥?/sup> and OCS. A previous theoretical study suggested that salt bridges between Arg297 and Glu20 or Asp111 initiated conformational changes critical for catalysis. However, our MD simulations and steady-state kinetic experiments did not corroborate this result. Similar k_cat/K_m values for both the E20A and D111A SsuD variants to wild-type SsuD suggest that the salt bridges are not critical to the desulfonation mechanism. Instead, the predicted role of Arg297 is to favorably interact with the phosphate group of the reduced flavin. Concomitantly, Arg226 functioned as a 鈥減rotection鈥?group shielding FMNOO鈥?/sup> from bulk solvent and was more pronounced when both FMNOO鈥?/sup> and OCS were bound. The stabilization of FMNOO鈥?/sup> through electrostatic interactions with Arg226 would properly position the C4a peroxy group for the proposed nucleophilic attack on the sulfur of octanesulfonate.}