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Identification of the Flavin Binding Site in the Flavin Reductase from the Alkanesulfonate Monooxygenase System in Escherichia coli
Author(s) -
Sun Honglei,
Gao Benlian,
Ellis Holly
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb53-d
Subject(s) - flavin group , escherichia coli , chemistry , biochemistry , stereochemistry , enzyme kinetics , active site , monooxygenase , enzyme , gene , cytochrome p450
Sulfate starvation in Escherichia coli increases the synthesis of specific proteins which utilize alternate sulfur sources for growth. A tow‐component enzyme system has been show to be involved in the conversion of alkanesulfonates to sulfides and the corresponding aldehyde. The system consists of two proteins, a flavin reductase (SsuE) and a monooxygenase (SsuD) which together catalyze the oxidation of alkanesulfonate to the products. Previous studies have identified a conserved amino acid sequence, RXFS, from an alignment search of E. coli flavin reductases. The Ser residue has been shown to be involved in flavin binding by interacting with the isoalloxazine ring at N(5). An alignment search of SsuE also identified a similar conserved amino acid sequence motif, R 51 FDS 54 , which may be involved in flavin binding. Ser54 from wild type SsuE was substituted with alanine and the SsuES54A protein purified. Results from circular dichroism spectroscopy of SsuES54A were essentially identical to wild type SsuE suggesing there were no major secondary structural changes due to this mutation. The k m and k cat values for SsuES54A were 0.09 μM and 101.83 min −1 respectively. Compared with previous results from wild type SsuE, the k m increased by 6‐fold and the catalytic efficiency k cat /k m decreased by 6.4‐fold. In addition, the different k d value for SsuES54A compared to the wild type protein suggested this mutation altered the affinity of flavin binding. Our results show that Ser54 plays an essential role in flavin binding to SsuE. To further probe the flavin binding sites, studies involving multiple mutations of the conserved R 51 FDS 54 region will be performed. This research is supported by Auburn University.