Fig. 4: Enzymatic production of FAla in vitro and a workflow to determine the enantiomeric excess.

a The scheme indicates the enzymatic cascade assembled for FAla synthesis coupled with the regeneration of the reduced cofactor [NAD(P)H]. The enzyme pair ^StDAPDH and NADP-^PseFDH uses FPyr and formate as substrates; FPyr is converted into (S)-FAla by ^StDAPDH, whereas NADPH is regenerated by formate oxidation to CO₂. The equivalent reactions are catalyzed by the enzyme pair ^VpALD and NAD-^PseFDH, in this case forming (R)-FAla and providing NADH. b Chiral purity is assessed after the reaction mixture is derivatized with the Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, FDAA), which converts the FAla enantiomers into non-enantiomeric derivatives that can be separated by reversed-phase chromatography. c Representative HPLC profiles showing a reaction blank, a mixture of authentic (S)-FAla and (R)-FAla standards, a sample of the reaction containing ^StDAPDH and a sample with ^VpALD. mA.U., arbitrary units × 10^–3. d Quantification of the FAla formation in terms of FPyr conversion and enantiomeric excess, ee, for ^VpALD and ^StDAPDH. The graph represents mean values and the error bars correspond to standard deviations from three independent experiments; individual data points are indicated in the plot. N.D., not detected.