21-27636-ARTICLEO 13 H 7 15 16 3 HO 1 five OH 9 OH 7 O 13 H CCR9 Source 15a2221 21 + no enzb12 1 9 5 H H 7 H HO 1 three HO 5 OH 9 H12 16 H12 H21 21 + SptF 24 24 + no enz3 HOandrosterone (21)R 13 H 7 15 16 three O 512 9 OH 7 H 13 H 15 OH25-24 24 + SptF 28 28+ no enz3 O9 HH25 testosterone (24): R = OH progesterone (28): R = COCH12 1 three 9 H 5 O 7 H H 13 15 OH 16 three O 1 9 H five OH 7 12 H 13 H 15 OH30, 3128 28+ SptF+ m/z 291.two for (21) + m/z 289.2 for (24) + m/z 315.2 for (28)+ m/z 287.2 for (22) + m/z 305.2 for (25) + m/z 313.2 for (29)+ m/z 323.two for (23) + m/z 303.2 for (26, 27) + m/z 331.two for (30, 31)OFig. three Oxidation of human steroid hormones by SptF. a LC-MS EICs for in vitro assays applying androsterone (21), testosterone (24), and progesterone (28). The + m/z value employed for each and every compound is shown. b Structures of substrates and corresponding products. Merchandise 22, 23, and 257 were isolated from massive scale enzymatic reactions, and also the structures of 22, 23, and 26/27 were determined by NMR, when the structure of 25 was solved by the crystal sponge process. Note that 26 and 27 are interchangeable with one another based on the NMR data. Products 291 were not determined because of separation complications, but their structures have been deduced to become hydroxylated and/or oxidized compounds, thinking about their molecular weights (Supplementary Fig. 2).kcat/KM worth of F133A for 1 decreased more than 1000-fold as compared with the wild-type enzyme (Supplementary Table 4). F133A also converted 1 into the C11-hydroxylated item 33 (Supplementary Figs. 89-94 and Supplementary Table 20). In contrast, the substitution of ErbB3/HER3 list Phe133 with Tyr retained the generation of both four and 5 (Fig. 6b). Furthermore, the I231A variant showed a similar LC-MS profile to that of F133Y, although affording 32 as the major item. The loss on the sec-butyl group increases the active website space around the D-ring of 1, which could cause an option substrate binding mode. Notably, similar final results have been observed in the enzyme reactions together with the unnatural substrate 15. The activity for the generation from the cyclopropane ring was abolished in F133A, but retained in F133Y. Additionally, I231A drastically decreased the activity to produce 16 (Fig. 6c), suggesting the similar role in the hydrophobic residues within the recognition with the unnatural substrate 15. Compound 33, generated by F133A variant, is really a proposed biosynthetic intermediate of two (Supplementary Fig. 1)40. On the other hand, in vitro enzyme reaction of 33 with SptF revealed that 33 is just not converted for the final products 3, and thus 33 is just not an intermediate inside the pathway (Supplementary Fig. 13). Secondly, the hydrophilic residues that interact with substrates, including Asn65, Ser114, Thr148, and Asn150, were substituted with alanine (Fig. 6a-c). On account of the insolubility of T148A, the T148S variant was made use of for the enzyme reaction. As a result, N65A substantially reduced the four and five formation activity to 20 and accumulated 3 (Fig. 6b, e), suggesting that the hydrogenbond interaction with Asn65 just isn’t entirely crucial but moderately critical for the binding of 3. Interestingly, as compared with wild-type SptF, the activities for the formation of 4 and 5 have been maintained at 83 , 93 , and 52 in the S114A, T148S, and N150A variants, respectively. The ratio of 4 and 5 wasaltered to 7:3 in S114A and T148S, as compared with four:6 inside the wild-type, and decreased to 1:9 in N150A (Fig. 6e). Our observations suggested that these 3 residues ascertain the position of th