Rational Design of the Spatial Effect in a Fe(II)/α-Ketoglutarate-Dependent Dioxygenase Reverses the Regioselectivity of C(sp3)-H Bond Hydroxylation in Aliphatic Amino Acids.

The hydroxylation of remote C(sp3)-H bonds in aliphatic amino acids yields crucial precursors for the synthesis of high-value compounds. However, accurate regulation of the regioselectivity of remote C(sp3)-H bonds hydroxylation in aliphatic amino acids continues to be a common challenge in chemosynthesis and biosynthesis. In this study, the Fe(II)/α-ketoglutarate-dependent dioxygenase from Bacillus subtilis (BlAH) was mined and found to catalyze hydroxylation at the γ and δ sites of aliphatic amino acids. Crystal structure analysis, molecular dynamics simulations, and quantum chemical calculations revealed that regioselectivity was regulated by the spatial effect of BlAH. Based on these results, the spatial effect of BlAH was reconstructed to stabilize the transition state at the δ site of aliphatic amino acids, thereby successfully reversing the γ site regioselectivity to the δ site. For example, the regioselectivity of L-Homoleucine (5 a) was reversed from the γ site (1 : 12) to the δ site (>99 : 1). The present study not only expands the toolbox of biocatalysts for the regioselective functionalization of remote C(sp3)-H bonds, but also provides a theoretical guidance for the precision-driven modification of similarly remote C(sp3)-H bonds in complex molecules.

Efficient Production of Epoxy-Norbornane from Norbornene by an Engineered P450 Peroxygenase.

Epoxy-norbornane (EPO-NBE) is a crucial building block for the synthesis of various biologically active heterocyclic systems. To develop an efficient protocol for producing EPO-NBE using norbornene (NBE) as a substrate, cytochrome P450 enzyme from Pseudomonas putida (CYP238A1) was examined and its crystal structure (PDB code: 7X53) was resolved. Molecular mechanism analysis showed a high energy barrier related to iron-alkoxy radical complex formation. Therefore, a protein engineering strategy was developed and an optimal CYP238A1NPV variant containing a local hydrophobic "fence" at the active site was obtained, which increased the H2 O2 -dependent epoxidation activity by 7.5-fold compared with that of CYP238A1WT . Among the "fence", Glu255 participates in an efficient proton transfer system. Whole-cell transformation using CYP238A1NPV achieved an EPO-NBE yield of 77.6 g ⋅ L-1 in a 30-L reactor with 66.3 % conversion. These results demonstrate the potential of this system for industrial production of EPO-NBE and provides a new biocatalytic platform for epoxidation chemistry.

Combined 3D-QSAR, molecular docking and molecular dynamics study on the benzimidazole inhibitors targeting HCV NS5B polymerase.

The hepatitis C virus (HCV)-infected population has continued to grow during recent years, and novel HCV antiviral agents are urgently needed. In this work, a combined theoretical study was performed on the HCV non-structural 5B (NS5B) polymerase and 53 benzimidazole inhibitors. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were carried out with ligand-based and receptor-based alignments. Ligand-based QSAR models (cross-validated q2 of 0.918 for CoMFA and 0.825 for CoMSIA) were found to be superior to receptor-based approaches (cross-validated q2 of 0.765 for CoMFA and 0.740 for CoMSIA). Based on the most predictive CoMFA and CoMSIA models, the structural features that were essential for the inhibitory activity of benzimidazoles were characterized. A molecular dynamics study revealed that the induced fit effect between NS5B and its substrate may be responsible for the inferiority of the receptor-based CoMFA and CoMSIA models. The binding-free energy calculated using the MM/PBSA method correlated well with the experimental results and revealed that the van der Waals and electrostatic interactions most contributed to the binding. In addition, energetically favorable NS5B residues were identified by the per-residue decomposition of binding-free energy. The results presented in this work provide meaningful information for the design of novel benzimidazole inhibitors targeting the NS5B polymerase.Communicated by Ramaswamy H. Sarma.