Fig. 3: Bioinformatic and experimental analysis identify that flavone reductases (FLRs) are a distinct group of ene-reductases.

a Phylogenetic analysis of FLRs. The maximum-likelihood phylogenetic tree is based on the protein sequences of putative FLR enzymes found in Ascomycota, Firmicutes, Spirochetes, Fusobacteria, and Actinobacteria. Light purple circles on branches indicate bootstrap values greater than 0.7 from 300 bootstrap replicates. The FLR-like enzymes highlighted with red stars were chosen to assay the in vitro activity towards the flavone apigenin. b A sequence similarity network (SSN) for the ene-reductases. The network was generated with an initial score of 10⁻²⁰ and the “alignment score threshold” (that is a measure of the minimum sequence similarity threshold) for drawing the edges that connect the proteins (nodes) in the SSN was then refined such that nodes are connected by an edge if this value is ≥40. Each of the nodes represents proteins with ≥90% amino acid sequence identity and was colored according to the cluster type (OYE, EnoR, SDR, MDR, QnoR, and FLR). Each type of ene-reductase, as shown in a different color, is separated into different clusters that may contain enzymes with similar biochemical activity. A total of 72 different FLR-like proteins which are shown in the phylogenic tree are denoted in red. The nodes representing the reported ene-reductase with enzyme activity data (from BRENDA Enzyme Database) and biochemically identified FLR-like enzymes in this study were highlighted with lemon yellow. OYE, Old-Yellow-Enzyme; EnoR, oxygen-sensitive enoate reductases; SDR, short-chain dehydrogenase/reductase; MDR, medium-chain dehydrogenase/reductase; QnoR, quinone reductase-like ene-reductase.