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Kaoru Saigo - One of the best experts on this subject based on the ideXlab platform.
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structure and site directed mutagenesis of a flavoprotein from escherichia coli that reduces nitrocompounds alteration of pyridine nucleotide binding by a single amino acid substitution
Journal of Biological Chemistry, 2001Co-Authors: Toshiro Kobori, Shuhei Zenno, Kaoru Saigo, Hiroshi Sasaki, Michael E P Murphy, Masaru TanokuraAbstract:Abstract The crystal structure of a major oxygen-insensitive nitroReductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an α + β-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent Flavin Reductase ofVibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with theV. harveyi Flavin Reductase and the NADPH-dependent nitro/Flavin Reductase of Bacillus subtilis, residues Arg203 and Arg208 of the loop region between helices I and J in the vicinity of the calalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in theK m value for NADPH indicating that the side chain of Arg203 plays a key role in binding NADPH possibly to interact with the 2′-phosphate group.
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Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution.
The Journal of biological chemistry, 2000Co-Authors: Toshiro Kobori, Shuhei Zenno, Kaoru Saigo, Woo Cheol Lee, Hiroshi Sasaki, Michael E P Murphy, Masaru TanokuraAbstract:The crystal structure of a major oxygen-insensitive nitroReductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent Flavin Reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi Flavin Reductase and the NADPH-dependent nitro/Flavin Reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.
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Conversion of NfsA, the major Escherichia coli nitroReductase, to a Flavin Reductase with an activity similar to that of Frp, a Flavin Reductase in Vibrio harveyi, by a single amino acid substitution.
Journal of bacteriology, 1998Co-Authors: Shuhei Zenno, Masaru Tanokura, Toshiro Kobori, Kaoru SaigoAbstract:NfsA is the major oxygen-insensitive nitroReductase of Escherichia coli, similar in amino acid sequence to Frp, a Flavin Reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroReductase into a Flavin Reductase that is as active as the authentic Frp and a tartrazine Reductase that is 30-fold more active than wild-type NfsA.
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Gene Cloning, Purification, and Characterization of NfsB, a Minor Oxygen-Insensitive NitroReductase from Escherichia coli, Similar in Biochemical Properties to FRase I, the Major Flavin Reductase in Vibrio Fischeri
Journal of biochemistry, 1996Co-Authors: Shuhei Zenno, Hideaki Koike, Masaru Tanokura, Kaoru SaigoAbstract:nfsB, encoding a minor oxygen-insensitive nitroReductase, was isolated by PCR using primers corresponding to two amino acid sequences conserved among the major Flavin Reductase from Vibrio fischeri and classical nitroReductases from Salmonella typhimurium and Enterobacter cloacae. The gene product, NfsB, was purified to homogeneity from extracts of Escherichia coli cells overexpressing it. NfsB was found to be situated at 13 min on the E. coli map. Biochemical analysis indicated NfsB to be a polypeptide having a calculated molecular weight of 23,904, capable of forming a homodimer and associated tightly with FMN as a prosthetic group. Although it exhibited a lower affinity to the NfsB apoenzyme than FMN, FAD could serve as an effective substitute for FMN. It was also shown that NfsB has a broad electron acceptor specificity and is associated with a low level of the NAD(P)H-Flavin oxidoReductase. The NfsB catalysis obeys the ping pong Bi-Bi mechanism. The Km value for NADH varied depending on the second substrate used.
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Conversion of NfsB, a minor Escherichia coli nitroReductase, to a Flavin Reductase similar in biochemical properties to FRase I, the major Flavin Reductase in Vibrio fischeri, by a single amino acid substitution.
Journal of bacteriology, 1996Co-Authors: Shuhei Zenno, Hideaki Koike, Masaru Tanokura, Kaoru SaigoAbstract:NfsB is an oxygen-insensitive nitroReductase of Escherichia coli with significant amino acid sequence homology to the major Flavin Reductase (FRase I) of Vibrio fischeri. Here, we show that NfsB is convertible to an FRase I-like Flavin Reductase three times as active as the authentic FRase I by a single amino acid substitution in the least-conserved region.
Shuhei Zenno - One of the best experts on this subject based on the ideXlab platform.
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structure and site directed mutagenesis of a flavoprotein from escherichia coli that reduces nitrocompounds alteration of pyridine nucleotide binding by a single amino acid substitution
Journal of Biological Chemistry, 2001Co-Authors: Toshiro Kobori, Shuhei Zenno, Kaoru Saigo, Hiroshi Sasaki, Michael E P Murphy, Masaru TanokuraAbstract:Abstract The crystal structure of a major oxygen-insensitive nitroReductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an α + β-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent Flavin Reductase ofVibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with theV. harveyi Flavin Reductase and the NADPH-dependent nitro/Flavin Reductase of Bacillus subtilis, residues Arg203 and Arg208 of the loop region between helices I and J in the vicinity of the calalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in theK m value for NADPH indicating that the side chain of Arg203 plays a key role in binding NADPH possibly to interact with the 2′-phosphate group.
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Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution.
The Journal of biological chemistry, 2000Co-Authors: Toshiro Kobori, Shuhei Zenno, Kaoru Saigo, Woo Cheol Lee, Hiroshi Sasaki, Michael E P Murphy, Masaru TanokuraAbstract:The crystal structure of a major oxygen-insensitive nitroReductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent Flavin Reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi Flavin Reductase and the NADPH-dependent nitro/Flavin Reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.
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Conversion of NfsA, the major Escherichia coli nitroReductase, to a Flavin Reductase with an activity similar to that of Frp, a Flavin Reductase in Vibrio harveyi, by a single amino acid substitution.
Journal of bacteriology, 1998Co-Authors: Shuhei Zenno, Masaru Tanokura, Toshiro Kobori, Kaoru SaigoAbstract:NfsA is the major oxygen-insensitive nitroReductase of Escherichia coli, similar in amino acid sequence to Frp, a Flavin Reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroReductase into a Flavin Reductase that is as active as the authentic Frp and a tartrazine Reductase that is 30-fold more active than wild-type NfsA.
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Gene Cloning, Purification, and Characterization of NfsB, a Minor Oxygen-Insensitive NitroReductase from Escherichia coli, Similar in Biochemical Properties to FRase I, the Major Flavin Reductase in Vibrio Fischeri
Journal of biochemistry, 1996Co-Authors: Shuhei Zenno, Hideaki Koike, Masaru Tanokura, Kaoru SaigoAbstract:nfsB, encoding a minor oxygen-insensitive nitroReductase, was isolated by PCR using primers corresponding to two amino acid sequences conserved among the major Flavin Reductase from Vibrio fischeri and classical nitroReductases from Salmonella typhimurium and Enterobacter cloacae. The gene product, NfsB, was purified to homogeneity from extracts of Escherichia coli cells overexpressing it. NfsB was found to be situated at 13 min on the E. coli map. Biochemical analysis indicated NfsB to be a polypeptide having a calculated molecular weight of 23,904, capable of forming a homodimer and associated tightly with FMN as a prosthetic group. Although it exhibited a lower affinity to the NfsB apoenzyme than FMN, FAD could serve as an effective substitute for FMN. It was also shown that NfsB has a broad electron acceptor specificity and is associated with a low level of the NAD(P)H-Flavin oxidoReductase. The NfsB catalysis obeys the ping pong Bi-Bi mechanism. The Km value for NADH varied depending on the second substrate used.
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Conversion of NfsB, a minor Escherichia coli nitroReductase, to a Flavin Reductase similar in biochemical properties to FRase I, the major Flavin Reductase in Vibrio fischeri, by a single amino acid substitution.
Journal of bacteriology, 1996Co-Authors: Shuhei Zenno, Hideaki Koike, Masaru Tanokura, Kaoru SaigoAbstract:NfsB is an oxygen-insensitive nitroReductase of Escherichia coli with significant amino acid sequence homology to the major Flavin Reductase (FRase I) of Vibrio fischeri. Here, we show that NfsB is convertible to an FRase I-like Flavin Reductase three times as active as the authentic FRase I by a single amino acid substitution in the least-conserved region.
Masaru Tanokura - One of the best experts on this subject based on the ideXlab platform.
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Crystal Structures of the Short-Chain Flavin Reductase HpaC from Sulfolobus tokodaii Strain 7 in Its Three States: NAD(P)+-Free, NAD+-Bound, and NADP+-Bound†,‡
Biochemistry, 2006Co-Authors: Masahiko Okai, Norio Kudo, Woo Cheol Lee, Masayuki Kamo, Koji Nagata, Masaru TanokuraAbstract:4-Hydroxyphenylacetate (4-HPA) is oxidized as an energy source by two component enzymes, the large component (HpaB) and the small component (HpaC). HpaB is a 4-HPA monooxygenase that utilizes FADH2 supplied by a Flavin Reductase HpaC. We determined the crystal structure of HpaC (ST0723) from the aerobic thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 in its three states [NAD(P)+-free, NAD+-bound, and NADP+-bound]. HpaC exists as a homodimer, and each monomer was found to contain an FMN. HpaC preferred FMN to FAD because there was not enough space to accommodate the AMP moiety of FAD in its Flavin-binding site. The most striking difference between the NAD(P)+-free and the NAD+/NADP+-bound structures was observed in the N-terminal helix. The N-terminal helices in the NAD+/NADP+-bound structures rotated ca. 20° relative to the NAD(P)+-free structure. The bound NAD+ has a compact folded conformation with nearly parallel stacking rings of nicotinamide and adenine. The nicotinamide of NAD+ stacked t...
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structure and site directed mutagenesis of a flavoprotein from escherichia coli that reduces nitrocompounds alteration of pyridine nucleotide binding by a single amino acid substitution
Journal of Biological Chemistry, 2001Co-Authors: Toshiro Kobori, Shuhei Zenno, Kaoru Saigo, Hiroshi Sasaki, Michael E P Murphy, Masaru TanokuraAbstract:Abstract The crystal structure of a major oxygen-insensitive nitroReductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an α + β-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent Flavin Reductase ofVibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with theV. harveyi Flavin Reductase and the NADPH-dependent nitro/Flavin Reductase of Bacillus subtilis, residues Arg203 and Arg208 of the loop region between helices I and J in the vicinity of the calalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in theK m value for NADPH indicating that the side chain of Arg203 plays a key role in binding NADPH possibly to interact with the 2′-phosphate group.
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Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution.
The Journal of biological chemistry, 2000Co-Authors: Toshiro Kobori, Shuhei Zenno, Kaoru Saigo, Woo Cheol Lee, Hiroshi Sasaki, Michael E P Murphy, Masaru TanokuraAbstract:The crystal structure of a major oxygen-insensitive nitroReductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent Flavin Reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi Flavin Reductase and the NADPH-dependent nitro/Flavin Reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.
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Conversion of NfsA, the major Escherichia coli nitroReductase, to a Flavin Reductase with an activity similar to that of Frp, a Flavin Reductase in Vibrio harveyi, by a single amino acid substitution.
Journal of bacteriology, 1998Co-Authors: Shuhei Zenno, Masaru Tanokura, Toshiro Kobori, Kaoru SaigoAbstract:NfsA is the major oxygen-insensitive nitroReductase of Escherichia coli, similar in amino acid sequence to Frp, a Flavin Reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroReductase into a Flavin Reductase that is as active as the authentic Frp and a tartrazine Reductase that is 30-fold more active than wild-type NfsA.
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Gene Cloning, Purification, and Characterization of NfsB, a Minor Oxygen-Insensitive NitroReductase from Escherichia coli, Similar in Biochemical Properties to FRase I, the Major Flavin Reductase in Vibrio Fischeri
Journal of biochemistry, 1996Co-Authors: Shuhei Zenno, Hideaki Koike, Masaru Tanokura, Kaoru SaigoAbstract:nfsB, encoding a minor oxygen-insensitive nitroReductase, was isolated by PCR using primers corresponding to two amino acid sequences conserved among the major Flavin Reductase from Vibrio fischeri and classical nitroReductases from Salmonella typhimurium and Enterobacter cloacae. The gene product, NfsB, was purified to homogeneity from extracts of Escherichia coli cells overexpressing it. NfsB was found to be situated at 13 min on the E. coli map. Biochemical analysis indicated NfsB to be a polypeptide having a calculated molecular weight of 23,904, capable of forming a homodimer and associated tightly with FMN as a prosthetic group. Although it exhibited a lower affinity to the NfsB apoenzyme than FMN, FAD could serve as an effective substitute for FMN. It was also shown that NfsB has a broad electron acceptor specificity and is associated with a low level of the NAD(P)H-Flavin oxidoReductase. The NfsB catalysis obeys the ping pong Bi-Bi mechanism. The Km value for NADH varied depending on the second substrate used.
Vincent Niviere - One of the best experts on this subject based on the ideXlab platform.
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The Flavin Reductase ActVB from Streptomyces coelicolor: characterization of the electron transferase activity of the flavoprotein form.
FEBS Letters, 2005Co-Authors: Laurent Filisetti, Marc Fontecave, Julien Valton, Vincent NiviereAbstract:The Flavin Reductase ActVB is involved in the last step of actinorhodin biosynthesis in Streptomyces coelicolor. Although ActVB can be isolated with some FMN bound, this form was not involved in the Flavin Reductase activity. By studying the ferric Reductase activity of ActVB, we show that its FMN-bound form exhibits a proper enzymatic activity of reduction of iron complexes by NADH. This shows that ActVB active site exhibits a dual property with regard to the FMN. It can use it as a substrate that goes in and off the active site or as a cofactor to provide an electron transferase activity to the polypeptide.
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Mechanism and substrate specificity of the Flavin Reductase ActVB from Streptomyces coelicolor.
Journal of Biological Chemistry, 2003Co-Authors: Laurent Filisetti, Marc Fontecave, Vincent NiviereAbstract:ActVB is the NADH:Flavin oxidoReductase participating in the last step of actinorhodin synthesis in Streptomyces coelicolor. It is the prototype of a whole class of Flavin Reductases with both sequence and functional similarities. The mechanism of reduction of free Flavins by ActVB has been studied. Although ActVB was isolated with FMN bound, we have demonstrated that it is not a flavoprotein. Instead, ActVB contains only one Flavin binding site, suitable for the Flavin Reductase activity and with a high affinity for FMN. In addition, ActVB proceeds by an ordered sequential mechanism, where NADH is the first substrate. Whereas ActVB is highly specific for NADH, it is able to catalyze the reduction of a great variety of natural and synthetic Flavins, but with K(m) values ranging from 1 microm (FMN) to 69 microm (lumiFlavin). We show that both the ribitol-phosphate chain and the isoalloxazine ring contribute to the protein-Flavin interaction. Such properties are unique and set the ActVB family apart from the well characterized Fre Flavin Reductase family.
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Crystal structure of NAD(P)H:Flavin oxidoReductase from Escherichia coli.
Biochemistry, 1999Co-Authors: Margareta Ingelman, Marc Fontecave, Vincent Niviere, S. Ramaswamy, Hans EklundAbstract:Flavin Reductases use Flavins as substrates and are distinct from flavoenzymes which have tightly bound Flavins. The reduced Flavin can serve to reduce ferric complexes and iron proteins. In Escherichia coli, reactivation of ribonucleotide Reductase is achieved by reduced Flavins produced by Flavin Reductase. The crystal structure of E. coli Flavin Reductase reveals that the enzyme structure is similar to the structures of the ferredoxin Reductase family of flavoproteins despite very low sequence similarities. The main difference between Flavin Reductase and structurally related flavoproteins is that there is no binding site for the AMP moiety of FAD. The direction of the helix in the Flavin binding domain, corresponding to the phosphate binding helix in the flavoproteins, is also slightly different and less suitable for phosphate binding. Interactions for Flavin substrates are instead provided by a hydrophobic isoalloxazine binding site that also contains a serine and a threonine, which form hydrogen bon...
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An original electroenzymatic system: Flavin Reductase-riboFlavin for the improvement of dehydrogenase-based biosensors. Application to the amperometric detection of lactate
Electroanalysis, 1997Co-Authors: S Cosnier, Marc Fontecave, Christophe Innocent, Vincent NiviereAbstract:The electropolymerization of a Flavin Reductase (Fre) - amphiphilic pyrrole ammonium mixture previously adsorbed on the electrode surface provides an efficient bioelectrode for the amperometric detection of NADH and NADPH. The bioelectrode response was based on the oxidation at -0.1 V vs. SCE of the enzymically generated dihydroriboFlavin. The sensitivity and detection limit of the bioelectrode were 29 mAM-' cm-' and 0.2pM for NADH and €5.8mAM-' cm-' and 0.4pM for NADPH. The coimmobilization of Fre and a lactate dehydrogenase leads to a dehydrogenase-based biozlectrode for the determination of lactate in the presence of riboFlavin and NAD'. With laponite additives into the polypyrrole host matrix, the sensitivity and the detection limit of the bioelectrode for lactate are 11.7 m.4M-I cm? and 1 pM respectively. Owing to the negative value of the applied potential for the oxidation of riboFlavin, the response of the bienzyme electrode remains insensitive to interferents like ascorbate, urate and acetaminophen.
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An original electroenzymatic system: Flavin Reductase-riboFlavin for the improvement of dehydrogenase-based biosensors. Application to the amperometric detection of lactate
Electroanalysis, 1997Co-Authors: S Cosnier, Marc Fontecave, Christophe Innocent, Vincent NiviereAbstract:The electropolymerization of a Flavin Reductase (Fre) – amphiphilic pyrrole ammonium mixture previously adsorbed on the electrode surface provides an efficient bioelectrode for the amperometric detection of NADH and NADPH. The bioelectrode response was based on the oxidation at −;0.1 V vs. SCE of the enzymically generated dihydroriboFlavin. The sensitivity and detection limit of the bioelectrode were 29 mAM−1 cm−2 and 0.2 μM for NADH and 15.8 mAM−1 cm−2 and 0.4 μM for NADPH. The coimmobilization of Fre and a lactate dehydrogenase leads to a dehydrogenase-based bioelectrode for the determination of lactate in the presence of riboFlavin and NAD+. With laponite additives into the polypyrrole host matrix, the sensitivity and the detection limit of the bioelectrode for lactate are 11.7 mAM−1 cm−2 and 1 μM respectively. Owing to the negative value of the applied potential for the oxidation of riboFlavin, the response of the bienzyme electrode remains insensitive to interferents like ascorbate, urate and acetaminophen.
Toshiro Kobori - One of the best experts on this subject based on the ideXlab platform.
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structure and site directed mutagenesis of a flavoprotein from escherichia coli that reduces nitrocompounds alteration of pyridine nucleotide binding by a single amino acid substitution
Journal of Biological Chemistry, 2001Co-Authors: Toshiro Kobori, Shuhei Zenno, Kaoru Saigo, Hiroshi Sasaki, Michael E P Murphy, Masaru TanokuraAbstract:Abstract The crystal structure of a major oxygen-insensitive nitroReductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an α + β-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent Flavin Reductase ofVibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with theV. harveyi Flavin Reductase and the NADPH-dependent nitro/Flavin Reductase of Bacillus subtilis, residues Arg203 and Arg208 of the loop region between helices I and J in the vicinity of the calalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in theK m value for NADPH indicating that the side chain of Arg203 plays a key role in binding NADPH possibly to interact with the 2′-phosphate group.
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Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution.
The Journal of biological chemistry, 2000Co-Authors: Toshiro Kobori, Shuhei Zenno, Kaoru Saigo, Woo Cheol Lee, Hiroshi Sasaki, Michael E P Murphy, Masaru TanokuraAbstract:The crystal structure of a major oxygen-insensitive nitroReductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent Flavin Reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi Flavin Reductase and the NADPH-dependent nitro/Flavin Reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.
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Conversion of NfsA, the major Escherichia coli nitroReductase, to a Flavin Reductase with an activity similar to that of Frp, a Flavin Reductase in Vibrio harveyi, by a single amino acid substitution.
Journal of bacteriology, 1998Co-Authors: Shuhei Zenno, Masaru Tanokura, Toshiro Kobori, Kaoru SaigoAbstract:NfsA is the major oxygen-insensitive nitroReductase of Escherichia coli, similar in amino acid sequence to Frp, a Flavin Reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroReductase into a Flavin Reductase that is as active as the authentic Frp and a tartrazine Reductase that is 30-fold more active than wild-type NfsA.