Translational misreading, amino acid misincorporation and misinterpretations. The case of the flavocytochrome b2 H373Q variant

•Amino acid misincorporation occurs during protein biosynthesis.•Misincorporation in recombinant proteins can lead to errors in interpretations.•Two different expression systems for an Fcb2 variant both lead to misincorporation.•Missing the phenomenon led to an erroneous mechanistic interpretation.•Taking the phenomenon into account supports in this case a carbanion mechanism.

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.

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