Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

Abstract

Terpenoid natural products comprise a wide range of molecular architectures that typically result from C–C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

Two-Component FAD-Dependent Monooxygenases: Current Knowledge and Biotechnological Opportunities

“…Group D flavoprotein monooxygenases ( Table 1 ) catalyze hydroxylation of aromatic compounds but also N -hydroxylation reactions are described. Members of group D can be divided into FAD- and FMN-dependent enzymes, while some are also promiscuous towards the flavin co-substrate [ 7 , 48 , 49 ]. This clustering is also reflected by phylogenetic analysis of group D ( Figure 2 ).…”

“…Two of the first characterized members are the phenol 2-hydroxylase and the 4-hydroxyphenylacetate monooxygenase, which will be described in the first place to outline a general overview about FAD-dependent group D monooxygenases. Many group D monooxygenases that originate from Streptomyces are involved in the biosynthesis of secondary metabolites (antibiotics, antitumor agents) [ 48 , 49 , 60 , 68 ]. For example, a recently discovered enzyme (XiaF) is involved in the biosynthesis of an indolosesquiterpene (xiamycin) [ 48 ].…”

“…Many group D monooxygenases that originate from Streptomyces are involved in the biosynthesis of secondary metabolites (antibiotics, antitumor agents) [ 48 , 49 , 60 , 68 ]. For example, a recently discovered enzyme (XiaF) is involved in the biosynthesis of an indolosesquiterpene (xiamycin) [ 48 ]. Other recently characterized members are involved in the degradation and detoxification of halogenated phenols [ 58 , 59 ].…”

“…As already mentioned, the loop region in the middle domain can influence flavin as well as substrate preference but potentially also the reactivity. For instance, the hydroxylase XiaF shows hydroxylation-induced terpenoid cyclization [ 48 ]. The active site of XiaF is more open what might help to accommodate bigger substrates and absence of the loop that is required for recognition of the adenine domain might explain the promiscuity towards the flavin co-substrate ( Figure 5 ).…”

“…XiaF is displayed including surface (deep teal) with bound FAD (orange). The loop region and therefore the substrate-binding pocket (indicated by an arrow) is more open in XiaF, allowing for promiscuity towards the flavin co-substrate [ 48 ].…”