The electrochemical leaf enables the electrification and control of multi-enzyme cascades by exploiting two discoveries: (i) the ability to electrify the photosynthetic enzyme ferredoxin NADP+ reductase (FNR), driving it to catalyse the interconversion of NADP+/NADPH whilst it is entrapped in a highly porous, metal oxide electrode, and (ii) the evidence that additional enzymes can be co-entrapped in the electrode pores where, through one NADP(H)-dependent enzyme, extended cascades can be driven by electrical connection to FNR, via NADP(H) recycling. By changing a critical active-site tyrosine to serine, FNR's exclusivity for NADP(H) is swapped for unphosphorylated NAD(H). Here we present an electrochemical study of this variant FNR, and show that in addition to the intended inversion of cofactor preference, this change to the active site has altered FNR's tuning of the flavin reduction potential, making it less reductive. Exploiting the ability to monitor the variant's activity with NADP(H) as a function of potential has revealed a trapped intermediate state, relieved only by applying a negative overpotential, which allows catalysis to proceed. Inhibition by NADP+ (very tightly bound) with respect to NAD(H) turnover was also revealed and interestingly, this inhibition changes depending on the applied potential. These findings are of critical importance for future exploitation of the electrochemical leaf.
The prenylated-flavin mononucleotide-dependent decarboxylases (also known as UbiD-like enzymes) are the most recently discovered family of decarboxylases. The modified flavin facilitates the decarboxylation of unsaturated carboxylic acids through a novel mechanism involving 1,3-dipolar cyclo-addition chemistry. UbiD-like enzymes have attracted considerable interest for biocatalysis applications due to their ability to catalyse (de)carboxylation reactions on a broad range of aromatic substrates at otherwise unreactive carbon centres. There are now ∼35 000 protein sequences annotated as hypothetical UbiD-like enzymes. Sequence similarity network analyses of the UbiD protein family suggests that there are likely dozens of distinct decarboxylase enzymes represented within this family. Furthermore, many of the enzymes so far characterized can decarboxylate a broad range of substrates. Here we describe a strategy to identify potential substrates of UbiD-like enzymes based on detecting enzyme-catalysed solvent deuterium exchange into potential substrates. Using ferulic acid decarboxylase (FDC) as a model system, we tested a diverse range of aromatic and heterocyclic molecules for their ability to undergo enzyme-catalysed H/D exchange in deuterated buffer. We found that FDC catalyses H/D exchange, albeit at generally very low levels, into a wide range of small, aromatic molecules that have little resemblance to its physiological substrate. In contrast, the sub-set of aromatic carboxylic acids that are substrates for FDC-catalysed decarboxylation is much smaller. We discuss the implications of these findings for screening uncharacterized UbiD-like enzymes for novel (de)carboxylase activity.