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Robert C. Bray - One of the best experts on this subject based on the ideXlab platform.
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Properties of Xanthine Dehydrogenase Variants from Rosy Mutant Strains of Drosophila melanogaster and their Relevance to the enzyme's Structure and Mechanism
European journal of biochemistry, 1996Co-Authors: Wendy A. Doyle, Julian F. Burke, Arthur Chovnick, F. Lee Dutton, J. Robert S. Whittle, Robert C. BrayAbstract:Xanthine Dehydrogenase, a molybdenum, iron-sulfur flavoenzyme encoded in the fruit fly Drosophila melanogaster by the rosy gene, has been characterised both from the wild-type and mutant flies. Enzyme assays, using a variety of different oxidising and reducing substrates were supplemented by limited molecular characterisation. Four rosy strains showed no detectable activity in any enzyme assay tried, whereas from four wild-type and three rosy mutant strains, those for the [E89K], [L127F] and [L157P]Xanthine Dehydrogenases (in all of which the mutation is in the iron-sulfur domain), the enzyme molecules, although present at different levels, had extremely similar or identical properties. This was confirmed by purification of one wild-type and one mutant enzyme, [E89K]Xanthine Dehydrogenase. These both had ultraviolet–visible absorption spectra similar to milk Xanthine oxidase. Both were found to be quite stable molecules, showing very high catalytic-centre activities and with little tendency to become degraded by proteolysis or modified by conversion to oxidase or desulfo forms. In three further rosy strains, giving [G353D]Xanthine Dehydrogenase and [S357F]Xanthine Dehydrogenase mutated in the flavin domain, and [G1011E]Xanthine Dehydrogenase mutated in the molybdenum domain, enzyme activities were selectively diminished in certain assays. For the G353D and S357F mutant enzymes activities to NAD+ as oxidising substrate were diminished, to zero for the latter. In addition for [G353D]Xanthine Dehydrogenase, there was an increase in apparent Km values both for NAD+ and NADH. These findings indicate involvement of this part of the sequence in the NAD+-binding site. The G1011E mutation has a profound effect on the enzyme. As isolated and as present in crude extracts of the flies, this Xanthine Dehydrogenase variant lacks activity to Xanthine or pterin as reducing substrate, indicating an impairment of the functioning of its molybdenum centre. However, it retains full activity to NADH with dyes as oxidising substrate. Mild oxidation of the enzyme converts it, apparently irreversibly, to a form showing full activity to Xanthine and pterin. The nature of the group that is oxidised is discussed in the light of redox potential data. It is proposed that the process involves oxidation of the pterin of the molybdenum cofactor from the tetrahydro to a dihydro oxidation state. This conclusion is fully consistent with recent information [Romao, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P. & Huber, R. (1995) Science 270, 1170–1176] from X-ray crystallography on the structure of a closely related enzyme from Desulfovibrio gigas. It is proposed, that apparent irreversibility of the oxidative activating process for [G1011E]Xanthine Dehydrogenase, is due to conversion of its pterin to the tricyclic derivative detected by these workers. The data thus provide the strongest evidence available, that the oxidation state of the pterin can have a controlling influence on the activity of a molybdenum cofactor enzyme. Implications regarding pterin incorporation into Xanthine Dehydrogenase and in relation to other molybdenum enzymes are discussed.
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Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of Xanthine Dehydrogenase.
Biochemical Journal, 1992Co-Authors: Richard K. Hughes, Wendy A. Doyle, Julian F. Burke, Arthur Chovnick, J. Robert S. Whittle, Robert C. BrayAbstract:The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the Xanthine Dehydrogenase sequence. Sequence comparisons permit identification, with some certainty, of regions associated with the iron-sulphur centres and the pterin molybdenum cofactor of the enzyme. Procedures have been developed and rigorously tested for the assay in gel-filtered extracts of the flies, of different catalytic activities of Xanthine Dehydrogenase by the use of various oxidizing and reducing substrates. These methods have been applied to 11 different rosy mutant strains that map to different regions of the sequence. All the mutations studied cause characteristic activity changes in the enzyme. In general these are consistent with the accepted assignment of the cofactors to the different domains and with the known reactivities of the molybdenum, flavin and iron-sulphur centres. Most results are interpretable in terms of the mutation affecting electron transfer to or from one redox centre only. The activity data provide evidence that FAD and the NAD+/NADH binding sites are retained in mutants mapping to the flavin domain. Therefore, despite some indications from sequence comparisons, it is concluded that the structure of this domain of Xanthine Dehydrogenase cannot be directly related to that of other flavoproteins for which structural data are available. The data also indicate that the artificial electron acceptor phenazine methosulphate acts at the iron-sulphur centres and suggest that these centres may not be essential for electron transfer between molybdenum and flavin. The work emphasizes the importance of combined genetic and biochemical study of rosy mutant Xanthine Dehydrogenase variants in probing the structure and function of enzymes of this class.
Kimiyoshi Ichida - One of the best experts on this subject based on the ideXlab platform.
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Investigation of the transport of Xanthine Dehydrogenase inhibitors by the urate transporter ABCG2.
Drug metabolism and pharmacokinetics, 2017Co-Authors: Makiko Nakamura, Kyoko Fujita, Yu Toyoda, Tappei Takada, Hiroshi Hasegawa, Kimiyoshi IchidaAbstract:Abstract Hyperuricemia induces gout and kidney stones and accelerates the progression of renal and cardiovascular diseases. Adenosine 5′-triphosphate-binding cassette subfamily G member 2 (ABCG2) is a urate transporter, and common dysfunctional variants of ABCG2, non-functional Q126X (rs72552713) and semi-functional Q141K (rs2231142), are risk factors for hyperuricemia and gout. A recent genome wide association study suggested that allopurinol, a serum uric acid-lowering drug that inhibits Xanthine Dehydrogenase, is a potent substrate of ABCG2. In this study, we aimed to examine the transport of Xanthine Dehydrogenase inhibitors via ABCG2. Our results show that ABCG2 transports oxypurinol, an active metabolite of allopurinol, whereas allopurinol and febuxostat, a new Xanthine Dehydrogenase inhibitor, are not substrates of ABCG2. The amount of oxypurinol transported by ABCG2 vesicles significantly increased in the presence of ATP, compared to that observed with mock vesicles. Since the half-life of oxypurinol is longer than that of allopurinol, the Xanthine Dehydrogenase-inhibiting effect of allopurinol mainly depends on its metabolite, oxypurinol. Our results indicate that the serum level of oxypurinol would increase in patients with ABCG2 dysfunction.
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MUTATIONS IN Xanthine Dehydrogenase GENE IN SUBJECTS WITH HEREDITARY XANTHINURIA
Advances in experimental medicine and biology, 1998Co-Authors: Kimiyoshi Ichida, Naoyuki Kamatani, Takeshi Nishino, Masakatsu Saji, Hideaki Okabe, Tatsuo HosoyaAbstract:Xanthine Dehydrogenase is an enzyme that catalyzes the oxidation of hypoXanthine to Xanthine and uric acid from Xanthine during the final stage of purine metabolism. Xanthine Dehydrogenase exists as a dimer and each subunit has a molecular weight of 145000. Human Xanthine Dehydrogenase cDNA consists of 4002 nucleotides (1) and the gene is mapped to chromosome 2p23 (2). Mapping of the functions on Xanthine Dehydrogenase was performed for 3 peptide domains generated by the protein cleavage (1). The N-terminal 20 kD domain includes a 2Fe/2S non-heme iron binding site while the adjacent 40 kD and the C-terminal 85 kD domains include flavin binding and molybdenum cofactor binding domains, respectively (1). Most of Xanthine Dehydrogenase usually exist as the Dehydrogenase form and the enzyme is converted to the oxidase form, Xanthine oxidase, by the proteolytic cleavage and the oxidation of cystein residues under certain conditions (3). Xanthine Dehydrogenase has recently been attracting attention for its possible involvement in triggering tissue damage by producing free radicals. It is exhibited in many studies that Xanthine oxidase injured the tissues on the conditions, such as post-ischemic reperfusion tissue injury, adult respiratory distress syndrome and lung injury resulting from influenza virus infection (4–8).
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identification of two mutations in human Xanthine Dehydrogenase gene responsible for classical type i xanthinuria
Journal of Clinical Investigation, 1997Co-Authors: Kimiyoshi Ichida, Naoyuki Kamatani, Takeshi Nishino, Tatsuo Hosoya, Yoshihiro Amaya, Osamu SakaiAbstract:Hereditary xanthinuria is classified into three categories. Classical xanthinuria type I lacks only Xanthine Dehydrogenase activity, while type II and molybdenum cofactor deficiency also lack one or two additional enzyme activities. In the present study, we examined four individuals with classical xanthinuria to discover the cause of the enzyme deficiency at the molecular level. One subject had a C to T base substitution at nucleotide 682 that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 228. The duodenal mucosa from the subject had no Xanthine Dehydrogenase protein while the mRNA level was not reduced. The two subjects who were siblings with type I xanthinuria were homozygous concerning this mutation, while another subject was found to contain the same mutation in a heterozygous state. The last subject who was also with type I xanthinuria had a deletion of C at nucleotide 2567 in cDNA that should generate a termination codon from nucleotide 2783. This subject was homozygous for the mutation and the level of mRNA in the duodenal mucosa from the subject was not reduced. Thus, in three subjects with type I xanthinuria, the primary genetic defects were confirmed to be in the Xanthine Dehydrogenase gene.
Takeshi Nishino - One of the best experts on this subject based on the ideXlab platform.
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MUTATIONS IN Xanthine Dehydrogenase GENE IN SUBJECTS WITH HEREDITARY XANTHINURIA
Advances in experimental medicine and biology, 1998Co-Authors: Kimiyoshi Ichida, Naoyuki Kamatani, Takeshi Nishino, Masakatsu Saji, Hideaki Okabe, Tatsuo HosoyaAbstract:Xanthine Dehydrogenase is an enzyme that catalyzes the oxidation of hypoXanthine to Xanthine and uric acid from Xanthine during the final stage of purine metabolism. Xanthine Dehydrogenase exists as a dimer and each subunit has a molecular weight of 145000. Human Xanthine Dehydrogenase cDNA consists of 4002 nucleotides (1) and the gene is mapped to chromosome 2p23 (2). Mapping of the functions on Xanthine Dehydrogenase was performed for 3 peptide domains generated by the protein cleavage (1). The N-terminal 20 kD domain includes a 2Fe/2S non-heme iron binding site while the adjacent 40 kD and the C-terminal 85 kD domains include flavin binding and molybdenum cofactor binding domains, respectively (1). Most of Xanthine Dehydrogenase usually exist as the Dehydrogenase form and the enzyme is converted to the oxidase form, Xanthine oxidase, by the proteolytic cleavage and the oxidation of cystein residues under certain conditions (3). Xanthine Dehydrogenase has recently been attracting attention for its possible involvement in triggering tissue damage by producing free radicals. It is exhibited in many studies that Xanthine oxidase injured the tissues on the conditions, such as post-ischemic reperfusion tissue injury, adult respiratory distress syndrome and lung injury resulting from influenza virus infection (4–8).
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The mechanism of conversion of Xanthine Dehydrogenase to Xanthine oxidase
Oxygen Homeostasis and Its Dynamics, 1998Co-Authors: Takeshi Nishino, Ken Okamoto, Shigeko Nakanishi, Hiroyuki Hori, Tomoko NishinoAbstract:Xanthine Dehydrogenase and Xanthine oxidase are complex metalloflavoproteins that represent alternate forms of the same gene product. The cDNAs encoding the enzymes have been cloned from several sources, and structural information is becoming available. Using purified enzyme, comparative analyses between the two forms were attempted by spectroscopic and kinetics methods. The most significant difference between the two forms is the protein conformation around flavin adenine dinucleotide (FAD), which changes the redox potential of the flavin and the reactivity of FAD with the electron acceptors, nicotinamide adenine dinucleotide (NAD) and molecular oxygen. The flavin semiquinone is thermodynamically stable in Xanthine Dehydrogenase but is unstable in Xanthine oxidase. Detailed analyses by stopped-flow techniques suggest that the flavin semiquinone reacts with oxygen to form superoxide anion while the fully reduced flavin reacts to form hydrogen peroxide. Although Xanthine Dehydrogenase can produce greater amounts of superoxide anion than Xanthine oxidase during Xanthine oxygen reaction, it seems not to be physiologically significant in the cell, where excess NAD exists under normal conditions.
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identification of two mutations in human Xanthine Dehydrogenase gene responsible for classical type i xanthinuria
Journal of Clinical Investigation, 1997Co-Authors: Kimiyoshi Ichida, Naoyuki Kamatani, Takeshi Nishino, Tatsuo Hosoya, Yoshihiro Amaya, Osamu SakaiAbstract:Hereditary xanthinuria is classified into three categories. Classical xanthinuria type I lacks only Xanthine Dehydrogenase activity, while type II and molybdenum cofactor deficiency also lack one or two additional enzyme activities. In the present study, we examined four individuals with classical xanthinuria to discover the cause of the enzyme deficiency at the molecular level. One subject had a C to T base substitution at nucleotide 682 that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 228. The duodenal mucosa from the subject had no Xanthine Dehydrogenase protein while the mRNA level was not reduced. The two subjects who were siblings with type I xanthinuria were homozygous concerning this mutation, while another subject was found to contain the same mutation in a heterozygous state. The last subject who was also with type I xanthinuria had a deletion of C at nucleotide 2567 in cDNA that should generate a termination codon from nucleotide 2783. This subject was homozygous for the mutation and the level of mRNA in the duodenal mucosa from the subject was not reduced. Thus, in three subjects with type I xanthinuria, the primary genetic defects were confirmed to be in the Xanthine Dehydrogenase gene.
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A novel Xanthine Dehydrogenase inhibitor (BOF-4272).
Advances in experimental medicine and biology, 1991Co-Authors: Seiji Sato, Kunihiko Tatsumi, Takeshi NishinoAbstract:An inhibitor of Xanthine oxidase (XO)/Xanthine Dehydrogenase (XDH), an enzyme catalyzing the last step of purine catabolism, might be expected to be effective as remedy for hyperuricemia and possibly for ischemia-reperfusion injury. However, no clinically effective XO/XDH inhibitor except allopurinol have been used since it was introduced for clinical use in 1962 (1, 2).
Wendy A. Doyle - One of the best experts on this subject based on the ideXlab platform.
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Properties of Xanthine Dehydrogenase Variants from Rosy Mutant Strains of Drosophila melanogaster and their Relevance to the enzyme's Structure and Mechanism
European journal of biochemistry, 1996Co-Authors: Wendy A. Doyle, Julian F. Burke, Arthur Chovnick, F. Lee Dutton, J. Robert S. Whittle, Robert C. BrayAbstract:Xanthine Dehydrogenase, a molybdenum, iron-sulfur flavoenzyme encoded in the fruit fly Drosophila melanogaster by the rosy gene, has been characterised both from the wild-type and mutant flies. Enzyme assays, using a variety of different oxidising and reducing substrates were supplemented by limited molecular characterisation. Four rosy strains showed no detectable activity in any enzyme assay tried, whereas from four wild-type and three rosy mutant strains, those for the [E89K], [L127F] and [L157P]Xanthine Dehydrogenases (in all of which the mutation is in the iron-sulfur domain), the enzyme molecules, although present at different levels, had extremely similar or identical properties. This was confirmed by purification of one wild-type and one mutant enzyme, [E89K]Xanthine Dehydrogenase. These both had ultraviolet–visible absorption spectra similar to milk Xanthine oxidase. Both were found to be quite stable molecules, showing very high catalytic-centre activities and with little tendency to become degraded by proteolysis or modified by conversion to oxidase or desulfo forms. In three further rosy strains, giving [G353D]Xanthine Dehydrogenase and [S357F]Xanthine Dehydrogenase mutated in the flavin domain, and [G1011E]Xanthine Dehydrogenase mutated in the molybdenum domain, enzyme activities were selectively diminished in certain assays. For the G353D and S357F mutant enzymes activities to NAD+ as oxidising substrate were diminished, to zero for the latter. In addition for [G353D]Xanthine Dehydrogenase, there was an increase in apparent Km values both for NAD+ and NADH. These findings indicate involvement of this part of the sequence in the NAD+-binding site. The G1011E mutation has a profound effect on the enzyme. As isolated and as present in crude extracts of the flies, this Xanthine Dehydrogenase variant lacks activity to Xanthine or pterin as reducing substrate, indicating an impairment of the functioning of its molybdenum centre. However, it retains full activity to NADH with dyes as oxidising substrate. Mild oxidation of the enzyme converts it, apparently irreversibly, to a form showing full activity to Xanthine and pterin. The nature of the group that is oxidised is discussed in the light of redox potential data. It is proposed that the process involves oxidation of the pterin of the molybdenum cofactor from the tetrahydro to a dihydro oxidation state. This conclusion is fully consistent with recent information [Romao, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P. & Huber, R. (1995) Science 270, 1170–1176] from X-ray crystallography on the structure of a closely related enzyme from Desulfovibrio gigas. It is proposed, that apparent irreversibility of the oxidative activating process for [G1011E]Xanthine Dehydrogenase, is due to conversion of its pterin to the tricyclic derivative detected by these workers. The data thus provide the strongest evidence available, that the oxidation state of the pterin can have a controlling influence on the activity of a molybdenum cofactor enzyme. Implications regarding pterin incorporation into Xanthine Dehydrogenase and in relation to other molybdenum enzymes are discussed.
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Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of Xanthine Dehydrogenase.
Biochemical Journal, 1992Co-Authors: Richard K. Hughes, Wendy A. Doyle, Julian F. Burke, Arthur Chovnick, J. Robert S. Whittle, Robert C. BrayAbstract:The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the Xanthine Dehydrogenase sequence. Sequence comparisons permit identification, with some certainty, of regions associated with the iron-sulphur centres and the pterin molybdenum cofactor of the enzyme. Procedures have been developed and rigorously tested for the assay in gel-filtered extracts of the flies, of different catalytic activities of Xanthine Dehydrogenase by the use of various oxidizing and reducing substrates. These methods have been applied to 11 different rosy mutant strains that map to different regions of the sequence. All the mutations studied cause characteristic activity changes in the enzyme. In general these are consistent with the accepted assignment of the cofactors to the different domains and with the known reactivities of the molybdenum, flavin and iron-sulphur centres. Most results are interpretable in terms of the mutation affecting electron transfer to or from one redox centre only. The activity data provide evidence that FAD and the NAD+/NADH binding sites are retained in mutants mapping to the flavin domain. Therefore, despite some indications from sequence comparisons, it is concluded that the structure of this domain of Xanthine Dehydrogenase cannot be directly related to that of other flavoproteins for which structural data are available. The data also indicate that the artificial electron acceptor phenazine methosulphate acts at the iron-sulphur centres and suggest that these centres may not be essential for electron transfer between molybdenum and flavin. The work emphasizes the importance of combined genetic and biochemical study of rosy mutant Xanthine Dehydrogenase variants in probing the structure and function of enzymes of this class.
Julian F. Burke - One of the best experts on this subject based on the ideXlab platform.
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Properties of Xanthine Dehydrogenase Variants from Rosy Mutant Strains of Drosophila melanogaster and their Relevance to the enzyme's Structure and Mechanism
European journal of biochemistry, 1996Co-Authors: Wendy A. Doyle, Julian F. Burke, Arthur Chovnick, F. Lee Dutton, J. Robert S. Whittle, Robert C. BrayAbstract:Xanthine Dehydrogenase, a molybdenum, iron-sulfur flavoenzyme encoded in the fruit fly Drosophila melanogaster by the rosy gene, has been characterised both from the wild-type and mutant flies. Enzyme assays, using a variety of different oxidising and reducing substrates were supplemented by limited molecular characterisation. Four rosy strains showed no detectable activity in any enzyme assay tried, whereas from four wild-type and three rosy mutant strains, those for the [E89K], [L127F] and [L157P]Xanthine Dehydrogenases (in all of which the mutation is in the iron-sulfur domain), the enzyme molecules, although present at different levels, had extremely similar or identical properties. This was confirmed by purification of one wild-type and one mutant enzyme, [E89K]Xanthine Dehydrogenase. These both had ultraviolet–visible absorption spectra similar to milk Xanthine oxidase. Both were found to be quite stable molecules, showing very high catalytic-centre activities and with little tendency to become degraded by proteolysis or modified by conversion to oxidase or desulfo forms. In three further rosy strains, giving [G353D]Xanthine Dehydrogenase and [S357F]Xanthine Dehydrogenase mutated in the flavin domain, and [G1011E]Xanthine Dehydrogenase mutated in the molybdenum domain, enzyme activities were selectively diminished in certain assays. For the G353D and S357F mutant enzymes activities to NAD+ as oxidising substrate were diminished, to zero for the latter. In addition for [G353D]Xanthine Dehydrogenase, there was an increase in apparent Km values both for NAD+ and NADH. These findings indicate involvement of this part of the sequence in the NAD+-binding site. The G1011E mutation has a profound effect on the enzyme. As isolated and as present in crude extracts of the flies, this Xanthine Dehydrogenase variant lacks activity to Xanthine or pterin as reducing substrate, indicating an impairment of the functioning of its molybdenum centre. However, it retains full activity to NADH with dyes as oxidising substrate. Mild oxidation of the enzyme converts it, apparently irreversibly, to a form showing full activity to Xanthine and pterin. The nature of the group that is oxidised is discussed in the light of redox potential data. It is proposed that the process involves oxidation of the pterin of the molybdenum cofactor from the tetrahydro to a dihydro oxidation state. This conclusion is fully consistent with recent information [Romao, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P. & Huber, R. (1995) Science 270, 1170–1176] from X-ray crystallography on the structure of a closely related enzyme from Desulfovibrio gigas. It is proposed, that apparent irreversibility of the oxidative activating process for [G1011E]Xanthine Dehydrogenase, is due to conversion of its pterin to the tricyclic derivative detected by these workers. The data thus provide the strongest evidence available, that the oxidation state of the pterin can have a controlling influence on the activity of a molybdenum cofactor enzyme. Implications regarding pterin incorporation into Xanthine Dehydrogenase and in relation to other molybdenum enzymes are discussed.
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Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of Xanthine Dehydrogenase.
Biochemical Journal, 1992Co-Authors: Richard K. Hughes, Wendy A. Doyle, Julian F. Burke, Arthur Chovnick, J. Robert S. Whittle, Robert C. BrayAbstract:The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the Xanthine Dehydrogenase sequence. Sequence comparisons permit identification, with some certainty, of regions associated with the iron-sulphur centres and the pterin molybdenum cofactor of the enzyme. Procedures have been developed and rigorously tested for the assay in gel-filtered extracts of the flies, of different catalytic activities of Xanthine Dehydrogenase by the use of various oxidizing and reducing substrates. These methods have been applied to 11 different rosy mutant strains that map to different regions of the sequence. All the mutations studied cause characteristic activity changes in the enzyme. In general these are consistent with the accepted assignment of the cofactors to the different domains and with the known reactivities of the molybdenum, flavin and iron-sulphur centres. Most results are interpretable in terms of the mutation affecting electron transfer to or from one redox centre only. The activity data provide evidence that FAD and the NAD+/NADH binding sites are retained in mutants mapping to the flavin domain. Therefore, despite some indications from sequence comparisons, it is concluded that the structure of this domain of Xanthine Dehydrogenase cannot be directly related to that of other flavoproteins for which structural data are available. The data also indicate that the artificial electron acceptor phenazine methosulphate acts at the iron-sulphur centres and suggest that these centres may not be essential for electron transfer between molybdenum and flavin. The work emphasizes the importance of combined genetic and biochemical study of rosy mutant Xanthine Dehydrogenase variants in probing the structure and function of enzymes of this class.
