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Characterization of a Two-component Alkanesulfonate Monooxygenase from Escherichia coli

Eichhorn, Eric, van der Ploeg, Jan R., Leisinger, Thomas·1999·Journal of Biological Chemistry
DOI 10.1074/jbc.274.38.26639
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FIG. 1 Purification of the two components of the alkanesulfonate monooxygenase system from E. coli . Protein samples (30 m g) were analyzed at different stages of purification on 12% SDS-PAGE gels under reducing and denaturing conditions, and stained with Coomassie Brilliant Blue. A , purification of NAD(P)H:FMN oxidoreductase SsuE. M, molecular weight markers (with molecular masses indicated on the left in kDa); lane 1, cell extract of uninduced E. coli BL21(DE3)(pME4287); lane 2, extract of cells producing wild type SsuE from pME4146; lane 3, extract of induced E. coli BL21(DE3)(pME4287) cells producing histidine-tagged SsuE; lane 4, wash with binding buffer; lane 5, wash with 60 mM imidazole; lane 6, purified SsuE. B , purification of alkanesulfonate monooxygenase SsuD. M, molecular weight markers (with molecular masses indicated on the left in kDa): lane 1, cell extract of uninduced E. coli BL21(DE3)(pME4282); lane 2, extract of induced cells; lane 3, fraction after Fractogel EMD SO 3 2 (S) chromatography; lane 4, fraction after Fractogel EMD TMAE (S) chromatography; lane 5, purified SsuD obtained after phenyl-Sepharose Fast Flow 66 chromatography.
FIG. 1 Purification of the two components of the alkanesulfonate monooxygenase system from E. coli . Protein samples (30 m g) were analyzed at different stages of purification on 12% SDS-PAGE gels under reducing and denaturing conditions, and stained with Coomassie Brilliant Blue. A , purification of NAD(P)H:FMN oxidoreductase SsuE. M, molecular weight markers (with molecular masses indicated on the left in kDa); lane 1, cell extract of uninduced E. coli BL21(DE3)(pME4287); lane 2, extract of cells producing wild type SsuE from pME4146; lane 3, extract of induced E. coli BL21(DE3)(pME4287) cells producing histidine-tagged SsuE; lane 4, wash with binding buffer; lane 5, wash with 60 mM imidazole; lane 6, purified SsuE. B , purification of alkanesulfonate monooxygenase SsuD. M, molecular weight markers (with molecular masses indicated on the left in kDa): lane 1, cell extract of uninduced E. coli BL21(DE3)(pME4282); lane 2, extract of induced cells; lane 3, fraction after Fractogel EMD SO 3 2 (S) chromatography; lane 4, fraction after Fractogel EMD TMAE (S) chromatography; lane 5, purified SsuD obtained after phenyl-Sepharose Fast Flow 66 chromatography.CC-BY · From the PDF
Image 2
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FIG. 2 Activity of NAD(P)H:FMN oxidoreductase SsuE in the presence of various flavin substrates. Activity measurements were performed under aerobic conditions as described under 'Experimental Procedures' in reaction mixtures containing 47 pmol of SsuE, 200 m M NADPH and varying concentrations of FMN, FAD, riboflavin, or lumiflavin in 20 mM imidazole, 50 mM NaCl, 20 mM Tris-HCl, pH 7.9. Initial rates were calculated from the slope of the absorbance decrease at 340 nm by regression analysis. Each point represents the mean of two independent experiments.
FIG. 2 Activity of NAD(P)H:FMN oxidoreductase SsuE in the presence of various flavin substrates. Activity measurements were performed under aerobic conditions as described under 'Experimental Procedures' in reaction mixtures containing 47 pmol of SsuE, 200 m M NADPH and varying concentrations of FMN, FAD, riboflavin, or lumiflavin in 20 mM imidazole, 50 mM NaCl, 20 mM Tris-HCl, pH 7.9. Initial rates were calculated from the slope of the absorbance decrease at 340 nm by regression analysis. Each point represents the mean of two independent experiments.CC-BY · From the PDF
FIG. 3 Influence of the molar ratio of the two components on alkanesulfonate desulfonation. The effect of the SsuE/SsuD molar ratio on alkanesulfonate desulfonation by SsuD was investigated by keeping the SsuD concentration constant at 55 nM and varying the concentration of SsuE in the reaction mixture described under 'Experimental Procedures.' Sulfite release was followed over 3 min and initial rates calculated from the slope of absorbance increase at 430 nm by regression analysis. Each point corresponds to the mean value of two independent experiments.
FIG. 3 Influence of the molar ratio of the two components on alkanesulfonate desulfonation. The effect of the SsuE/SsuD molar ratio on alkanesulfonate desulfonation by SsuD was investigated by keeping the SsuD concentration constant at 55 nM and varying the concentration of SsuE in the reaction mixture described under 'Experimental Procedures.' Sulfite release was followed over 3 min and initial rates calculated from the slope of absorbance increase at 430 nm by regression analysis. Each point corresponds to the mean value of two independent experiments.CC-BY · From the PDF
Image 5
CC-BY · From the PDF
FIG. 5 Alignment of SsuE with flavoprotein sequences. SsuE_ E. coli is NAD(P)H:FMN oxidoreductase from E. coli (Swiss-Prot accession number P80644), MsuE_Pseae is NADH:FMN oxidoreductase from Pseudomonas aeruginosa (TrEMBL accession number O31038), SsuE_Psepu is NADH:FMN oxidoreductase from Pseudomonas putida (TrEMBL accession number O85762) , ISF_Mette is iron-sulfur flavoprotein from M. thermophila (TrEMBL accession number Q50562), ISF_Metja and ISF2_Metja are putative iron-sulfur flavoproteins from Methanococcus janaschii (TrEMBL accession numbers Q58141 and Q58483). Sequences were aligned with the ClustalW program (30). Amino acid residues identical in at least three proteins are indicated in white on a black background. Amino acid residues conserved in at least three sequences are indicated in white on a gray background. Cysteine residues involved in the iron-sulfur cluster of flavoproteins are marked with an asterisk .
FIG. 5 Alignment of SsuE with flavoprotein sequences. SsuE_ E. coli is NAD(P)H:FMN oxidoreductase from E. coli (Swiss-Prot accession number P80644), MsuE_Pseae is NADH:FMN oxidoreductase from Pseudomonas aeruginosa (TrEMBL accession number O31038), SsuE_Psepu is NADH:FMN oxidoreductase from Pseudomonas putida (TrEMBL accession number O85762) , ISF_Mette is iron-sulfur flavoprotein from M. thermophila (TrEMBL accession number Q50562), ISF_Metja and ISF2_Metja are putative iron-sulfur flavoproteins from Methanococcus janaschii (TrEMBL accession numbers Q58141 and Q58483). Sequences were aligned with the ClustalW program (30). Amino acid residues identical in at least three proteins are indicated in white on a black background. Amino acid residues conserved in at least three sequences are indicated in white on a gray background. Cysteine residues involved in the iron-sulfur cluster of flavoproteins are marked with an asterisk .CC-BY · From the PDF
FIG. 6 Alignment of SsuD with the flavin-dependent enzymes Mer and LuxB. The following sequences were aligned with the program ClustalW (30): SsuD from E. coli (Swiss-Prot accession number P80645), NtaA from C. heintzii (Swiss-Prot accession number P54989), SnaA from S. pristinaespiralis (Swiss-Prot accession number P54991), SoxA from Rhodococcus sp. strain IGTS8 (Swiss-Prot accession number P54995), Mer from M. thermoautotrophicum (TrEMBL accession number Q50744), and LuxB and LuxA from Vibrio harveyi (Swiss-Prot accession numbers P07739 and P07740). Only the alignment between SsuD, Mer, and LuxB is shown. Identical residues in all three sequences are in white on a black background, identical residues occurring in two sequences are in white on a gray background. The secondary structure elements of LuxB (27) are shown below the sequence as arrows ( a -helices) or thick lines ( b -sheets). The predicted secondary structure elements of SsuD and Mer were calculated with the program PredictProtein (31) and are indicated by h for a -helices, e for b -sheets, and l for loops. Dots represent lack of defined secondary structure.
FIG. 6 Alignment of SsuD with the flavin-dependent enzymes Mer and LuxB. The following sequences were aligned with the program ClustalW (30): SsuD from E. coli (Swiss-Prot accession number P80645), NtaA from C. heintzii (Swiss-Prot accession number P54989), SnaA from S. pristinaespiralis (Swiss-Prot accession number P54991), SoxA from Rhodococcus sp. strain IGTS8 (Swiss-Prot accession number P54995), Mer from M. thermoautotrophicum (TrEMBL accession number Q50744), and LuxB and LuxA from Vibrio harveyi (Swiss-Prot accession numbers P07739 and P07740). Only the alignment between SsuD, Mer, and LuxB is shown. Identical residues in all three sequences are in white on a black background, identical residues occurring in two sequences are in white on a gray background. The secondary structure elements of LuxB (27) are shown below the sequence as arrows ( a -helices) or thick lines ( b -sheets). The predicted secondary structure elements of SsuD and Mer were calculated with the program PredictProtein (31) and are indicated by h for a -helices, e for b -sheets, and l for loops. Dots represent lack of defined secondary structure.CC-BY · From the PDF

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Metadata

Authors
Eichhorn, E., van der Ploeg, J. R., & Leisinger, T.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
1999-09
Volume
274
Issue
38
Pages
26639-26646
ISSN
0021-9258
DOI
10.1074/jbc.274.38.26639
Cited by
198
References
0
Language
en
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CoChat. (2026). Citation check: Characterization of a Two-component Alkanesulfonate Monooxygenase from Escherichia coli. https://app.cochat.ai/papers/10.1074/jbc.274.38.26639
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Eichhorn, E., van der Ploeg, J. R., & Leisinger, T. (1999). Characterization of a Two-component Alkanesulfonate Monooxygenase from Escherichia coli. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.274.38.26639