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Stehr, Matthias [VerfasserIn]; Smau, Liliana [VerfasserIn]; Singh, Mahavir [VerfasserIn]; Seth, Oliver [VerfasserIn]; Macheroux, Peter [VerfasserIn]; Ghisla, Sandro [VerfasserIn]; Diekmann, Hans [VerfasserIn]

Studies with lysine N6-hydroxylase. Effect of a mutation in the assumed FAD binding site on coenzyme affinities and on lysine hydroxylating activity - [published Version]

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  • Medientyp: E-Artikel; Sonstige Veröffentlichung
  • Titel: Studies with lysine N6-hydroxylase. Effect of a mutation in the assumed FAD binding site on coenzyme affinities and on lysine hydroxylating activity
  • Beteiligte: Stehr, Matthias [VerfasserIn]; Smau, Liliana [VerfasserIn]; Singh, Mahavir [VerfasserIn]; Seth, Oliver [VerfasserIn]; Macheroux, Peter [VerfasserIn]; Ghisla, Sandro [VerfasserIn]; Diekmann, Hans [VerfasserIn]
  • Erschienen: Berlin : De Gruyter, 1999
  • Erschienen in: Biological Chemistry 380 (1999), Nr. 1
  • Ausgabe: published Version
  • Sprache: Englisch
  • DOI: https://doi.org/10.15488/3157; https://doi.org/10.1515/BC.1999.006
  • ISSN: 1431-6730
  • Schlagwörter: nonhuman ; Lysine ; pH ; molecular genetics ; Binding Sites ; Genetic Vectors ; coenzyme ; amino acid substitution ; mixed function oxidase ; binding affinity ; genetics ; metabolism ; enzyme activity ; sequence homology ; enzyme structure ; mutant protein ; glycine ; Enzyme kinetics ; amino acid sequence ; Flavin-Adenine Dinucleotide ; site directed mutagenesis ; flavine adenine nucleotide ; binding site ; priority journal ; [...]
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  • Beschreibung: The proposed FAD binding site of L-lysine N6-hydroxylase (EC 1.14.13.99) exhibits an unusual proline in a position where a highly conserved glycine is found in other FAD dependent hydroxylases. We have studied the role of this proline by mutating it to glycine in [P14G]aerA, which was expressed in Escherichia coli M15-2 and purified to homogeneity. The mutation has marked effects on the affinities of the cofactors FAD and NADPH as well as the substrate, lysine. Compared to the wild-type enzyme, the activity vs. pH profile of the mutant protein indicates a shift of the apparent pK'(a)s (7.8 and 8.7 for wild-type and 6.8 and 7.7 for the P14G-mutant enzyme) and of the activity maximum (pH 8 for wild-type and pH 7 for the P14G-mutant enzyme). While the activity of the mutant enzyme is much lower under conditions found to be optimal for the wild-type enzyme, adjustment of substrate and cofactor concentrations and pH leads to comparable activities for the mutant enzyme. These results suggest that the proline fulfils an important structural role in the proposed FAD binding site.
  • Zugangsstatus: Freier Zugang