Ubiquitination is a type of post-translational modification wherein the small protein ubiquitin (Ub) is covalently bound to a lysine on a target protein. Ubiquitination can signal for several regulatory pathways including protein degradation. Ubiquitination occurs by a series of reactions catalyzed by three types of enzymes: ubiquitin activating enzymes, E1; ubiquitin conjugating enzymes, E2; and ubiquitin ligases, E3. E2 enzymes directly catalyze the transfer of Ub to the target protein�the RING E3 improves the efficiency. Prior to its transfer, Ub is covalently linked to the E2 via a thioester bond and the Ub∼E2 conjugate forms a quaternary complex with the RING E3. It is hypothesized that the RING E3 improves the catalytic efficiency of ubiquitination by placing the E2∼Ub conjugate in a "closed" position, which tensions and weakens the thioester bond. We interrogate this hypothesis by analyzing the strain on the thioester during molecular dynamics simulations of both open and closed E2∼Ub/E3 complexes. Our data indicate that the thioester is strained when the E2∼Ub conjugate is in the closed position. We also show that the amount of strain is consistent with the experimental rate enhancement caused by the RING E3. Finally, our simulations show that the closed configuration increases the populations of key hydrogen bonds in the E2∼Ub active site. This is consistent with another hypothesis stating that the RING E3 enhances reaction rates by preorganizing the substrates.

Ubiquitination is a type of post translational modification wherein the small protein ubiquitin (Ub) is covalently bound to a lysine on a target protein. Ubiquitination can signal for several regulatory pathways including protein degradation. Ubiquitination occurs by a series of reactions catalyzed by three types of enzymes: ubiquitin activating enzymes, E1; ubiquitin conjugating enzymes, E2; and ubiquitin ligases, E3. E2 enzymes directly catalyze the transfer of Ub to the target protein – the RING E3 improves the efficiency. Prior to its transfer, Ub is covalently linked to the E2 via a thioester bond and the Ub~E2 conjugate forms a quaternary complex with the RING E3. It is hypothesized that the RING E3 improves the catalytic efficiency of ubiquitination by placing the E2~Ub conjugate in a “closed” position, which tensions and weakens the thioester bond. We interrogate this hypothesis by analyzing the strain on the thioester during molecular dynamics simulations of both open and closed E2~Ub/E3 complexes. Our data indicate that the thioester is strained when the E2~Ub conjugate is in the closed position. We also show that the amount of strain is consistent with the experimental rate enhancement caused by the RING E3. Finally, our simulations show that the closed configuration increases the populations of key hydrogen bonds in the E2~Ub active site. This is consistent with another hypothesis stating that the RING E3 enhances reaction rates by preorganizing the substrates.

to androst-1,4-diene-3,17-dione (ADD) in much high-efficiency. However, the activity of KstDs is substrate-specific. In this study, we aimed to explore the effect of mycobacterial KstD (mKstD) structure on its substrate specificity. Based on protein modeling on mKstD in complex with 4-AD, we found that the residue Tyr116 (F116) in the active site of mKstD is adjacent to substrate and perturbed the orientation of the substrate. Thus, we explored how F116 affected enzymatic activity on different substrates with site-directed mutagenesis. Compared with the wild-type mKstD, the mutants F116V, F116A, F116G, F116W and F116L decreased the 4-AD conversion while the mutants F116I and F116Y significantly enhanced the bioconversion, indicating that F116 played a key role in the bioconversion from 4-AD to ADD. Our data also revealed that these F116 mutants exhibited different specificity for different steroidal substrates. We believed that our results would facilitate the manipulation of the catalytic properties of the enzyme to improve its application in the pharmaceutical steroid industry.