Photochemical Mechanism of Light-Driven Fatty Acid Photodecarboxylase

Abstract

Fatty acid photodecarboxylase (FAP) is a promising target for the production of biofuels and fine chemicals. It contains a flavin adenine dinucleotide cofactor and catalyzes the blue-light-dependent decarboxylation of fatty acids to generate the corresponding alkane. However, little is known about the catalytic mechanism of FAP, or how light is used to drive enzymatic decarboxylation. Here, we have used a combination of time-resolved and cryogenic trapping UV–visible absorption spectroscopy to characterize a red-shifted flavin intermediate observed in the catalytic cycle of FAP. We show that this intermediate can form below the “glass transition” temperature of proteins, whereas the subsequent decay of the species proceeds only at higher temperatures, implying a role for protein motions in the decay of the intermediate. Solvent isotope effect measurements, combined with analyses of selected site-directed variants of FAP, suggest that the formation of the red-shifted flavin species is directly coupled with hydrogen atom transfer from a nearby active site cysteine residue, yielding the final alkane product. Our study suggests that this cysteine residue forms a thiolate-flavin charge-transfer species, which is assigned as the red-shifted flavin intermediate. Taken together, our data provide insights into light-dependent decarboxylase mechanisms catalyzed by FAP and highlight important considerations in the (re)design of flavin-based photoenzymes.

Production of fatty alcohols from non-edible oils by enzymatic cascade reactions

“…As shown in Fig. 5 (and in Table S2, † entries [25][26][27] no signicant inuence of blue-light illumination was observed. In these experiments we also varied the reaction temperature and found only a minor inuence on the overall reaction rate.…”

“…Electron transfer from the avin to the thiyl radical and reprotonation closes the catalytic cycle (Scheme 2). 25 Very recently, we have reported the preparative application of CvFAP for the conversion of some (short and medium chain) carboxylic acids as well as for some natural fatty acids. [20][21][22][23][24] However, only edible oils and fatty acids have been considered so far, which we believe have only a limited potential for the preparation of biofuels.…”

Intensification of Photobiocatalytic Decarboxylation of Fatty Acids for the Production of Biodiesel

“…Therefore, we became very interested in the recently reported photoactivated fatty acid decarboxylase from Chlorella variabilis NC64A ( Cv FAP) [4] . Upon illumination with visible light ( λ =450 nm) Cv FAP catalyzes the decarboxylation of a broad range of fatty acids [4–15] . However, photochemical processes are severely limited by current reactor designs involving external illumination.…”

Promoter engineering for microbial bio-alkane gas production

“…Lower than expected correlations could occur when one or both of the ‘activities’ monitored are sub-optimal or absent. For example, photoinactivation or lack of flavin incorporation of CvFAP leads to inactive protein ( 32 , 33 ), while the C-terminal RFP domain may be fully active. Conversely, the RFP maturation rate may not keep pace with high protein expression rates in E. coli ( 34 ).…”