Tailoring enzymes involved in the biosynthesis of angucyclines contain latent context-dependent catalytic activities

Abstract

Comparison of homologous angucycline modification enzymes from five closely related Streptomyces pathways (pga, cab, jad, urd, lan) allowed us to deduce the biosynthetic steps responsible for the three alternative outcomes: gaudimycin C, dehydrorabelomycin, and 11-deoxylandomycinone. The C-12b-hydroxylated urdamycin and gaudimycin metabolites appear to be the ancestral representatives from which landomycins and jadomysins have evolved as a result of functional divergence of the ketoreductase LanV and hydroxylase JadH, respectively. Specifically, LanV has acquired affinity for an earlier biosynthetic intermediate resulting in a switch in biosynthetic order and lack of hydroxyls at C-4a and C-12b, whereas in JadH, C-4a/C-12b dehydration has evolved into an independent secondary function replacing C-12b hydroxylation. Importantly, the study reveals that many of the modification enzymes carry several alternative, hidden, or ancestral catalytic functions, which are strictly dependent on the biosynthetic context.

Figure 1

The proposed angucycline redox modification reactions and the structures of the metabolites in the native pga, cab, lan, urd, and jad pathways as determined in vitro. See also Figure S1.

Figure 2

Graphical Representation of the Gene Clusters pga, cab, urd, lan and jad under Study. The genes encoding for the enzymes involved in angucyclione modification are flavin-dependent oxygenases (black) and short-chain alcohol reductases/oxygenases (dark grey). The clusters also show genes involved with the aglycone biosynthesis (white), tailoring (jadG, light grey) and regulation (pgaY, light grey).

Figure 3

Representative HPLC Traces at 428 nm of the Native In Vitro Reactions with 2 as a Substrate. (A) The substrate 2, (B) UrdE/UrdMred product 5 (C) LanE/LanV product 8, (D) JadH product 7 (E) JadH production of 7 was not affected by the presence of SDRs. Spontaneous formation of 7 (Kallio et al., 2011) was avoided by ensuring quantitative conversion of 3 before metabolite extraction. See also Figure S2.

Figure 4

Consumption of 2 (■) and Formation of Products (○) in Reactions Carried Out to Completion at Different NADPH Concentrations. (A) A typical profile of a reaction producing the 12b-hydroxylated product 5. Characteristically the NADPH-dependent steps do not overlap, and the product is not formed before 2 has been depleted as in the CabE/CabV, PgaE/PgaM (Kallio et al., 2011) and UrdE/UrdMred in vitro reactions (B) A typical profile of a reaction producing 8. The two NADPH-dependent steps occur simultaneously and the product is formed already during the conversion of 2 as in the LanE/LanV in vitro reaction. See also Figure S3.

Figure 5

Representative HPLC Traces of Reaction Products Obtained with Isolated 3 as Substrate at 449nm. (A) LanE/LanV under anaerobic conditions yields 8, (B) LanE/LanV in the presence of O₂ yields 5, (C) JadH/CabV in the presence of O₂ yields 5 (D) JadH/UrdMred in the presence of O₂ yields 5 (E) JadH/LanV in the presence of O₂ yields 5. See also Figure S5.

Figure 6

Measured and Theoretical ECD Spectra of 5, 2 and 8. (A) Experimental and (B) predicted ECD spectrum for the 4aR,6S,12bS enantiomer of 5, (C) measured and (D) calculated ECD spectrum for the 4aR,12bR enantiomer of 2. The calculated spectrum was based on two molecules of 2 with one in a C-4-in conformation and the other in a C-4-out conformation, (E) experimental and (F) predicted spectrum for the 6R enantiomer of 8. Three molecules of 8 with two molecules having an equatorially disposed HO-6 group and the third containing an axially disposed HO-6 group were used in the calculations. See also Figure S4 and Table S1.