| Autor: |
Thakur A; Department of Chemistry, University of Miami, Coral Gables, Florida 33146, United States., Somai S; Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, United States., Yue K; Department of Chemistry, University of Miami, Coral Gables, Florida 33146, United States., Ippolito N; Department of Chemistry, University of Miami, Coral Gables, Florida 33146, United States., Pagan D; Department of Chemistry, University of Miami, Coral Gables, Florida 33146, United States., Xiong J; West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu 610041, China., Ellis HR; Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, United States., Acevedo O; Department of Chemistry, University of Miami, Coral Gables, Florida 33146, United States. |
| Abstrakt: |
Substrate-induced conformational changes present in alkanesulfonate monooxygenase (SsuD) are crucial to catalysis and lead to distinct interactions between a dynamic loop region and the active site. Accelerated molecular dynamics (aMD) simulations have been carried out to examine this potential correlation by studying wild-type SsuD and variant enzymes bound with different combinations of reduced flavin (FMNH 2 ), C4a-peroxyflavin intermediate (FMNOO - ), and octanesulfonate (OCS). Three distinct mobile loop conformations were identified: "open", "closed", and "semiclosed". The substrate-free SsuD system possessed a wide opening capable of providing full access for substrates to enter the active site. Upon binding FMNH 2 , SsuD adopts a closed conformation that would prevent unproductive oxidation reactions in the absence of OCS. Two salt bridges, Asp111-Arg263 and Glu205-Arg271, were identified as particularly important in maintaining the closed conformation. Experimental substitution of Arg271 to Ala did not alter the catalytic activity, but the variant in the presence of reduced flavin was more susceptible to proteolytic digestion compared to wild-type. With both FMNH 2 and OCS bound in SsuD, a second conformation was formed dependent upon a favorable π-π interaction between His124 and Phe261. Accordingly, there was no observed activity with the F261W SsuD variant in steady-state kinetic assays. This semiclosed conformation may be more appropriate for accepting O 2 into the binding pocket and/or may properly orient the active site for the ensuing oxygenolytic cleavage. Finally, simulations of SsuD simultaneously bound with FMNOO - and OCS found an open mobile loop region that suggests alternative flavin intermediates may participate in the reaction mechanism. |
