title: Deletional studies to investigate the functional role of a dynamic loop region of alkanesulfonate monooxygenase.
abstract: Several bacterial organisms rely on the two-component alkanesulfonate monooxygenase system for the acquisition of organosulfonate compounds when inorganic sulfur is limiting in the environment. This system is comprised of an FMN reductase (SsuE) that supplies reduced flavin to the alkanesulfonate monooxygenase (SsuD). Desulfonation of alkanesulfonates by SsuD is catalyzed through the activation of dioxygen by reduced flavin. The three-dimensional structure of SsuD exists as a TIM-barrel fold with several discrete insertion regions. An extensive insertion region near the putative active site was disordered in the SsuD structure, suggesting the importance of protein dynamics in the desulfonation mechanism. Three variants containing a partial deletion of the loop region were constructed to evaluate the functional properties of this region. There were no overall gross changes in secondary structure for the three SsuD deletion variants compared to wild-type SsuD, but each variant was found to be catalytically inactive. The deletion variants were unable to undergo the conformational changes necessary for catalysis even though they were able to bind reduced flavin. Rapid kinetic analyses monitoring the reductive and oxidative half-reactions indicated that the SsuD deletion variants failed to protect reduced flavin from unproductive oxidation. These studies define the importance of dynamic loop region for protection and stabilization of reduced flavin and reaction intermediates.
authors.affiliation: Department of Chemistry and Biochemistry, Auburn University, Auburn, AL, USA.
authors.forename: Jingyuan
authors.initials: J
authors.lastname: Xiong
authors.forename: Holly R
authors.initials: HR
authors.lastname: Ellis
mesh_terms: D000476:Alkanesulfonates / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D055162:Biocatalysis; D002384:Catalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D038181:FMN Reductase / Q000378:metabolism; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000476:Alkanesulfonates; D029968:Escherichia coli Proteins; D005415:Flavins; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C499579:SsuD protein, E coli; D038181:FMN Reductase; C447459:SsuE protein, E coli