Functional Evaluation of the π-Helix in the NAD(P)H:FMN Reductase of the Alkanesulfonate Monooxygenase System

Biochemistry ◽  
2018 ◽  
Vol 57 (30) ◽  
pp. 4469-4477 ◽  
Author(s):  
Jonathan M. Musila ◽  
Dianna L. Forbes ◽  
Holly R. Ellis
Keyword(s):  
Monooxygenase System ◽  
Functional Evaluation ◽  
Alkanesulfonate Monooxygenase

scholarly journals Detection of Protein-Protein Interactions in the Alkanesulfonate Monooxygenase System from Escherichia coli

2006 ◽  
Vol 188 (23) ◽  
pp. 8153-8159 ◽  
Author(s):  
Kholis Abdurachim ◽  
Holly R. Ellis
Keyword(s):  
Circular Dichroism ◽  
Protein Interactions ◽  
Visible Region ◽  
Circular Dichroism Spectroscopy ◽  
Flavin Mononucleotide ◽  
Stable Complex ◽  
Monooxygenase System ◽  
Protein Protein Interactions ◽  
Alkanesulfonate Monooxygenase ◽  
Circular Dichroïsm

ABSTRACT The two-component alkanesulfonate monooxygenase system utilizes reduced flavin as a substrate to catalyze a unique desulfonation reaction during times of sulfur starvation. The importance of protein-protein interactions in the mechanism of flavin transfer was analyzed in these studies. The results from affinity chromatography and cross-linking experiments support the formation of a stable complex between the flavin mononucleotide (FMN) reductase (SsuE) and monooxygenase (SsuD). Interactions between the two proteins do not lead to overall conformational changes in protein structure, as indicated by the results from circular dichroism spectroscopy in the far-UV region. However, subtle changes in the flavin environment of FMN-bound SsuE that occur in the presence of SsuD were identified by circular dichroism spectroscopy in the visible region. These data are supported by the results from fluorescent spectroscopy experiments, where a dissociation constant of 0.0022 ± 0.0010 μM was obtained for the binding of SsuE to SsuD. Based on these studies, the stoichiometry for protein-protein interactions is proposed to involve a 1:1 monomeric association of SsuE with SsuD.

scholarly journals Probing the flavin transfer mechanism in alkanesulfonate monooxygenase system

2018 ◽  
Author(s):  
PV Dayal ◽  
HR Ellis
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Lag Phase ◽  
Monooxygenase System ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Loop Region ◽  
Alkanesulfonate Monooxygenase ◽  
Α Helix

AbstractBacteria acquire sulfur through the sulfur assimilation pathway, but under sulfur limiting conditions bacteria must acquire sulfur from alternative sources. The alkanesulfonate monooxygenase enzymes are expressed under sulfur-limiting conditions, and catalyze the desulfonation of wide-range of alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase. The mechanism for the transfer of reduced flavin in flavin dependent two-component systems occurs either by free-diffusion or channeling. Previous studies have shown the presence of protein-protein interactions between SsuE and SsuD, but the identification of putative interaction sights have not been investigated. Current studies utilized HDX-MS to identify protective sites on SsuE and SsuD. A conserved α-helix on SsuD showed a decrease in percent deuteration when SsuE was included in the reaction. This suggests the role of α-helix in promoting protein-protein interactions. Specific SsuD variants were generated in order to investigate the role of these residues in protein-protein interactions and catalysis. Variant containing substitutions at the charged residues showed a six-fold decrease in the activity, while a deletion variant of SsuD lacking the α-helix showed no activity when compared to wild-type SsuD. In addition, there was no protein-protein interactions identified between SsuE and his-tagged SsuD variants in pull-down assays, which correlated with an increase in the Kd value. The α-helix is located right next to a dynamic loop region, positioned at the entrance of the active site. The putative interaction site and dynamic loop region located so close to the active site of SsuD suggests the importance of this region in the SsuD catalysis. Stopped-flow studies were performed to analyze the lag-phase which signifies the stabilization and transfer of reduced flavin from SsuE to SsuD. The SsuD variants showed a decrease in lag-phase, which could be because of a downturn in flavin transfer. A competitive assay was devised to evaluate the mechanism of flavin transfer in the alkanesulfonate monooxygenase system. A variant of SsuE was generated which interacted with SsuD, but was not able to reduce FMN. Assays that included varying concentrations of Y118A SsuE and wild-type SsuE in the coupled assays showed a decrease in the desulfonation activity of SsuD. The decrease in activity could be by virtue of Y118A SsuE competing with the wild-type SsuE for the putative docking site on SsuD. These studies define the importance of protein-protein interactions for the efficient transfer of reduced flavin from SsuE to SsuD leading to the desulfonation of alkanesulfonates.

scholarly journals Identifying the Mechanism of Reduced Flavin Transfer in Alkanesulfonate Monooxygenase System

2013 ◽  
Vol 27 (S1) ◽  
Author(s):  
Paritosh Vijay Dayal ◽  
Harsimran Singh ◽  
Laura Busenlehner ◽  
Holly Ellis
Keyword(s):  
Monooxygenase System ◽  
Alkanesulfonate Monooxygenase
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