AsqJ, an iron(II)- and 2-oxoglutarate-dependent enzyme found in viridicatin-type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3 epimer are reported. Meanwhile, a detailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short-lived Fe -oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate is preferred during AsqJ-catalyzed desaturation.

Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by non-heme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the non-heme iron-and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogs with different para substituents ranging from electron-donating groups (e.g. methoxy) to electronwithdrawing groups (e.g. trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, Mössbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations and molecular dynamic simulations collectively revealed the following mechanistic insights: 1) The rapid O 2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); 2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from non-heme Fe(IV)-oxo synthetic model complexes; 3) The OAT step most likely proceeds through a step-wise process with the initial formation of *

<div> <div> <div> <div> <p>One of the most important SARS-CoV-2 protein targets for therapeutics is the 3C-like protease (main protease, Mpro). In our previous work1​we used the first Mpro crystal structure to become available, 6LU7. On February 4, 2020 Insilico Medicine released the first potential novel protease inhibitors designed using a ​de novo,​AI-driven generative chemistry approach. Nearly 100 X-ray structures of Mpro co-crystallized both with covalent and non-covalent ligands have been published since then. Here we utilize the recently published 6W63 crystal structure of Mpro complexed with a non-covalent inhibitor and combined two approaches used in our previous study: ligand-based and crystal structure-based. We published 10 representative structures for potential development with 3D representation in PDB format and welcome medicinal chemists for broad discussion and generated output analysis. The molecules in SDF format and PDB-models for generated protein-ligand complexes are available here and at https://insilico.com/ncov-sprint/.​Medicinal chemistry VR analysis was provided by ​Nanome team and the video of VR session is available at ​https://bit.ly/ncov-vr.​ </p> </div> </div> </div> </div>

AsqJ,a ni ron(II)-and 2-oxoglutarate-dependent enzyme found in viridicatin-type alkaloid biosynthetic pathways,c atalyzess equential desaturation and epoxidation to produce cyclopenins.C rystal structures of AsqJ bound to cyclopeptin and its C3 epimer are reported. Meanwhile, ad etailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that apathway involving hydrogen atom abstraction at the C10 position of the substrate by as hort-lived Fe IV -oxo species and the subsequent formation of ac arbocation or ah ydroxylated intermediate is preferred during AsqJ-catalyzed desaturation.AsqJ,aniron(II) and 2-oxogluatrate (Fe/2OG) enzyme,has Scheme 1. A) AsqJ-catalyzed consecutivedesaturation and epoxidation. B) Examples of desaturation catalyzed by cytochrome P450 and di-iron cofactor enzymes.