Our bioinformatic analysis showed (KstDs from ReKstD1-like clade) that tyrosines of the proton relay system are not obligatory for KstD activity. Their substitution to phenylalanine results in a moderate decrease (2-3 fold) in the specific activity and slightly increases the barrier (TS2) of the hydride transfer to FAD. On the other hand, any mutation of tyrosines involved in catalysis leaves KstD either unable to activate the substrate (Y363F) or, while making the abstraction of the 2Hb proton easier, prohibitively increases the barrier of hydride transfer, rendering KstD virtually inactive.