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 overexpressed and characterized. SsuE was purified to homogeneity as an N-terminal histidine-tagged fusion protein. Native SsuE was a homodimeric enzyme of M(r) 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 pentanesulfonic acid to sulfite and pentaldehyde and was able to desulfonate a wide range of sulfonated substrates including C-2 to C-10 unsubstituted linear alkanesulfonates, substituted ethanesulfonic acids and sulfonated buffers. SsuD catalysis was absolutely dependent on FMNH(2) 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 FMNH(2)-dependent monooxygenase catalyzing the oxygenolytic conversion of alkanesulfonates to sulfite and the corresponding aldehydes. SsuE is the FMN reducing enzyme providing SsuD with FMNH(2).
Eichhorn
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E
Institut für Mikrobiologie, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich, Switzerland.
van der Ploeg
J R
JR
Leisinger
T
T
D000476:Alkanesulfonates / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D038181:FMN Reductase; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment
D000476:Alkanesulfonates; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases; C499579:SsuD protein, E coli; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases