9 by way of waters in N65T (shown in purple). Note that the coloring scheme for ligands would be the similar as in Fig. 4.We also tested the activities in the truncated variants with unnatural substrates, including the fungal meroterpenoids ten and 13, and steroids 21, 24, and 28. Consequently, each of the variants still maintained 240 and 564 activities toward 10 and 13, but dramatically decreased activities toward 21, 24, and 28 (20 ) (Fig. 6c, Supplementary Fig. 17, and Supplementary Table 6). These observations indicated that the lid-like loop region is essential for the steroid substrate recognition, but not necessary for the meroterpenoid substrates, that is consistent using the absence with the electron density on the loop in the complex structure with 15. The kinetics evaluation of the loop-truncation variant 9 revealed that the variants also maintained comparable activity toward 15 to that of wild-type (Supplementary Table four). Thus, the mutagenesis and kinetic assay outcomes strongly recommend that the malleability of your lid-like loop area HSF1 drug contributes to the outstanding substrate promiscuity and catalytic versatility of SptF. Discussions Substrate promiscuity plays a important part in both biomedical and industrial applications, because it delivers a starting point for protein engineering and drug design, enzyme evolution withdifferent reaction/substrate specificities, and chemoenzymatic total synthesis36,39,436. Hence, the identification and investigation of promiscuous enzymes enrich our understanding of how the enzymes get such a wide array of diverse functions, and can additional guide the rational engineering of those enzymes as biocatalysts for the production of beneficial molecules with improved biological activities. The Fe/KG oxygenase SptF exhibits uncommon promiscuity and catalytic versatility, and catalyzes four sequential oxidation reactions within the biosynthesis of fungal meroterpenoid emervaridones. SptF also catalyzes the formation of distinctive cyclopropane-ringfused, very congested 5/3/5/5/6/6 and 5/3/6/6/6 molecular scaffolds in the structurally distinct meroterpenoid terretonins. Additionally, SptF also hydroxylates steroids, which includes androsterone, testosterone, and progesterone. It is especially remarkable that SptF accepts each GLUT1 medchemexpress 3-keto-4- and 3-hydroxy-steroids with equivalent efficiencies to catalyze the hydroxylation in the -face of different positions and produce a series of hydroxylated steroids. Hydroxylation of steroids has been previously reported for cytochrome P450 enzymes47, which includes the human CYP3A subfamily plus the engineered bacterial P450 BM3, which catalyzesNATURE COMMUNICATIONS | (2022)13:95 | doi.org/10.1038/s41467-021-27636-3 | nature/naturecommunicationsNATURE COMMUNICATIONS | doi.org/10.1038/s41467-021-27636-ARTICLEO H6′ 2′ 7 8′ 4’H+ HO2 1 9O4′ 6′ 2′ 8’previously prososed pathway OH2H O HOOH HOOSptF-F133A FeIV O H2 1FeIII9′ 11 2’O3’4′ 6′ 8’OH 9′ SptF O2 1 9 3’O4′ 6’OH H O 4′ 9′ FeIII 3′ SptF2 11 9 1 2’O6′ 8’Fe IV O6′ 3′ 2′ 8’O4’FeIII O6′ 3′ 2′ 7 8’H14’SptFH12’H O HOOH H8’OOH O HOOH O HOO O HHOOandiconin D ( )emervaridoneB ( )O OO O3′ 8′ 6’O O3′ 8′ 6’H14’O O O OH14’SptF O OH O2 14′ 3’O6′ 8’FeIII O 4′ O O H1 9 3’Fe IV6′ 8’O H9 4′ 3′ 8’O O6’SptF O path a OOSptF O shunt path OH OH HOOH HOHO OH Fe IIIH H O Fe IVOO OH HOH7 HO H O FeIVO HOemervaridone C ( ) path b O H2 1 9 4’emeridone FSptF5’O O5’Fe III O5′ 16 O 7′ 6’OH4’HOH SptF HO4’O O2′ 7 5′ 16O 7′ 6’Fe IV O O H4’O5′ 6′ 7’O H O O H4′ 9 3’O2’6′ 7’3′ 2’16 O 6′ 7’H O HH8’2’2’OOOHO8’OH