was incubated in D2O buffer, the molecule weight of 11 doesn’t increase, which confirmed that the hydrogendeuterium exchange in 11 can’t be occurred (Supplementary Fig. 14a ). On the other hand, (two) when the AspoA-catalyzed isomerization of 7 to form 11 was instead performed in D2O buffer, the molecule weight in the generated 11 enhanced by 2 amu (m/z 388 [M + H]+, Supplementary Fig. 14a ), highly suggesting the DPP-2 Inhibitor site proposed dienol intermediate is indeed exist (Fig. 3b). (3) When the enzyme-prepared 2H-11 (m/z 388 [M + H]+) was incubated back to H2O buffer, the molecule weight on the 2H-11 will not reduce (Supplementary Fig. 14a ), which confirmed that these two deuteriums were incorporated into the nonactivated carbon atoms of 11, respectively (Supplementary Fig. 14c, e). (four) The 2H-11 was lastly prepared from the large-scale enzymatic conversionassays (SI), plus the subsequent 1H NMR analysis showed that these two deuteriums had been certainly incorporated into C19 and C20 of 11 (Supplementary Fig. 14d, e), respectively. (5) The spontaneous conversion of 7 to two in pH 4 D2O buffer confirmed that only one deuterium was incorporated into C20, while the incorporated deuterium was also not further wash-out for the duration of incubation of 2H-2 back to H2O buffer (Supplementary Fig. 15a ). The above each amino acid residues mutation and isotope labelling benefits confirmed that the AspoAcatalysed double bond isomerization contains protonation with the C21 carbonyl group, hydride shift and keto-enol tautomerization (Fig. 3b and Supplementary Fig. 14e). Despite the fact that these two conversions use the same precursors (7 and 8) and are all accomplished by way of protonation with the C21 carbonyl group (Fig. 3b), in comparison with the nonenzymatic conversion to form two and 1, AspoA strictly catalyses the production of 11 and 12. These results clearly suggest that the C13-C14 double bond, because the nucleophile to form the new C13-C19 bond, ought to beNATURE COMMUNICATIONS | (2022)13:225 | doi.org/10.1038/s41467-021-27931-z | nature/naturecommunicationsARTICLE14 12 13=210 nmNATURE COMMUNICATIONS | doi.org/10.1038/s41467-021-27931-zbiosynthesis and extremely recommend that the isolated pcCYTs and meCYTs are most likely artificially derived merchandise.AspoD+11+NADPHiMethodGeneral procedures. Reagents were purchased from Sigma-Aldrich, Thermo Fisher Scientific, or New England BioLabs. Primer synthesis and DNA sequencing had been performed by Sangon Biotech Co., Ltd. (Shanghai, China). The cIAP-1 Degrader manufacturer plasmids and primers used within this study are summarized in Supplementary Tables 1. All plasmids were extracted by the alkaline lysis strategy and dissolved in elution buffer. LC-MS analyses have been performed on a Waters ACQUITY H-Class UPLCMS method coupled to a PDA detector and an SQD2 mass spectrometer (MS) detector with an ESI supply. Chromatographic separation was performed at 35 using a C18 column (ACQUITY UPLCBEH, 1.7 m, 2.1 mm 100 mm, Waters). MPLC was performed on BUCHI RevelerisX2 Flash Chromatography System, with UV and ELSD detectors making use of BUCHI RevelerisC18 column (40 , 80 g). Semi-preparative HPLC was performed on Shimadzu Prominence HPLC system making use of a YMC-Pack ODS-A column (five m, 10 250 mm). MCI column chromatography (CC) was performed on an MCI gel CHP 20 P/P120 (375 m, Mitsubishi Chemical Corporation, Japan). NMR spectra had been recorded on a Bruker AVANCE III NMR (400 MHz) using a 5 mm broadband probe and TMS as an internal normal. HRMS data had been obtained on Fourier-transform ion cyclotron resonance-mass spectrometry (FT-ICR-MS) (
