title: Translational misreading, amino acid misincorporation and misinterpretations. The case of the flavocytochrome b2H373Q variant.
abstract: Amino acid misincorporation during protein synthesis occurs naturally at a low level. Protein sequence errors, depending on the level and the nature of the misincorporation, can have various consequences. When site-directed mutagenesis is used as a tool for understanding the role of a side chain in enzyme catalysis, misincorporation in a variant with intrinsically low activity may lead to misinterpretations concerning the enzyme mechanism. We report here one more example of such a problem, dealing with flavocytochrome b2(Fcb2), a lactate dehydrogenase, member of a family of FMN-dependent L-2-hydroxy acid oxidizing enzymes. Two papers have described the properties of the Fcb2 catalytic base H373Q variant, each one using a different expression system with the same base change for the mutation. The two papers found similar apparent kinetic parameters. But the first one demonstrated the existence of a low level of histidine misincorporation, which led to an important correction of the variant residual activity (Gaume et al. (1995) Biochimie, 77, 621). The second paper did not investigate the possibility of a misincorporation (Tsai et al. (2007) Biochemistry, 46, 7844). The two papers had different mechanistic conclusions. We show here that in this case the misincorporation does not depend on the expression system. We bring the proof that Tsai et al. (2007) were led to an erroneous mechanistic conclusion for having missed the phenomenon as well as for having misinterpreted the crystal structure of the variant. This work is another illustration of the caution one should exercise when characterizing enzyme variants with low activity.
authors.affiliation: Laboratoire de Chimie Physique, CNRS UMR 8000, Faculté des Sciences, Université Paris-Sud, Université Paris-Saclay, 91405 Orsay Cedex, France.
authors.forename: Mouna Ben
authors.initials: MB
authors.lastname: Elhoul
authors.affiliation: Laboratoire de Chimie Physique, CNRS UMR 8000, Faculté des Sciences, Université Paris-Sud, Université Paris-Saclay, 91405 Orsay Cedex, France.
authors.forename: Paul
authors.initials: P
authors.lastname: Machillot
authors.affiliation: Laboratoire de Chimie Physique, CNRS UMR 8000, Faculté des Sciences, Université Paris-Sud, Université Paris-Saclay, 91405 Orsay Cedex, France.
authors.forename: Mireille
authors.initials: M
authors.lastname: Benoît
authors.affiliation: Laboratoire de Chimie Physique, CNRS UMR 8000, Faculté des Sciences, Université Paris-Sud, Université Paris-Saclay, 91405 Orsay Cedex, France. Electronic address: florence.lederer@u-psud.fr.
authors.forename: Florence
authors.initials: F
authors.lastname: Lederer
mesh_terms: D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D042844:L-Lactate Dehydrogenase (Cytochrome) / Q000235:genetics* / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis / Q000235:genetics* / Q000502:physiology*
chemical_list: D000596:Amino Acids; D019344:Lactic Acid; D006639:Histidine; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)
keywords: Crystal structure; Enzyme mechanism; Flavocytochrome b(2); Flavoprotein; Misincorporation; Site-directed mutagenesis