Characterization of a two-component alkanesulfonate monooxygenase from Escherichia coli

The Escherichia coli ssuEADCB gene cluster is required for the utilization of alkanesulfonates as sulfur sources, and is expressed under conditions of sulfate or cysteine starvation. The SsuD and SsuE proteins were overexpres sed and characterized. SsuE was purified to homogeneity as an N-terminal hi stidine-tagged fusion protein. Native SsuE was a homodimeric enzyme of ill, 58,400, which catalyzed an NAD(P)H-dependent reduction of FMN, but it was also able to reduce FAD or riboflavin, The SsuD protein was purified to >98 % purity using cation exchange, anion exchange, and hydrophobic interaction chromatography. The pure enzyme catalyzed the conversion of pentanesulfoni c acid to sulfite and pentaldehyde and was able to desulfonate a wide range of sulfonated substrates including C-2 to C-10 unsubstituted linear alkane sulfonates, substituted ethanesulfonic acids and sulfonated buffers. SsuD c atalysis was absolutely dependent on FMNH2 and oxygen, and was maximal for SsuE/SsuD molar ratios of 2.1 to 4.2 in 10 mM Tris-HCl, pH 9.1. Native SsuD was a homotetrameric enzyme of M-r 181,000. These results demonstrate that SsuD is a broad range FMNH2-dependent monooxygenase catalyzing the oxygeno lytic conversion of alkanesulfonates to sulfite and the corresponding aldeh ydes, SsuE is the FMN reducing enzyme providing SsuD with FMNH2.