Conformational Dynamics in Corynebacterium glutamicum Diaminopimelate Epimerase: Insights from Ligand Parameterization, Atomistic Simulation, and Markov State Modeling.

The microbial enzyme diaminopimelate epimerase (DapF), a vital enzyme in the lysine biosynthetic pathway, catalyzes the conversion of L, L-diaminopimelate (L, L-DAP) to D, L-diaminopimelate (D, L-DAP) using a catalytic cysteine dyad with one cysteine in thiol state and another in thiolate. Under oxidizing conditions, the catalytic cysteines of apo DapF form a disulfide bond that alters the structure and function of DapF. Given its potential as a target for antimicrobial resistance treatments, understanding DapF's functional dynamics is imperative. In the present work, we employ microsecond-scale all-atom molecular dynamics simulations of product-bound DapF and apo-DapF under oxidized and reduced conditions. We employ a polarized charge model for the ligand and the active site residues, which was necessary to preserve the electrostatic environment in the active site and retain the ligand in the active site. The product-bound DapF and apo-DapF in oxidized and reduced conditions exhibit a closed, semi-open, and open conformation, respectively, as identified using the internal coordinates of the dimeric enzyme and the principal component analysis. The conformational switch is guided by the dynamic catalytic (DC) loop, loop II, and loop III movements in the active site. The time scale of the close-to-open conformational transition is estimated to be 0.8 µs through Markov state modeling (MSM) and transition path theory (TPT). The present study explains the role of various active site residues and loops in ligand binding and protein dynamics in the DapF enzyme under different redox conditions. Such information will be helpful in future inhibitor design studies targeting the DapF enzyme.

Functionalized Carbon Nano-Onions as a Smart Drug Delivery System for the Poorly Soluble Drug Carmustine for the Management of Glioblastoma.

The drug delivery system for transporting anticancer agents to targeted tissues in the body is a challenging issue. In search of a suitable biocompatible carrier having controlled and sustained drug release properties of poorly soluble drugs, carbon nano-onions (CNOs) were loaded with an anticancer drug, bis-chloroethyl nitrosourea (BCNU/carmustine). CNOs being autofluorescent, drug-loaded functionalized CNOs (f-CNO-BCNU) can be detected in vivo. Transmission electron microscopy (TEM) and differential light scattering (DLS) techniques were used to analyze the sizes of these f-CNOs. The molecular study revealed that the f-CNO-BCNU readily and noncovalently binds with the folate receptors present on the cancer cell surface in excess. Computer modeling and molecular dynamics simulation followed by binding free energy calculation shows f-CNOs have -29.9 kcal/mol binding free energy, and it noncovalently binds the receptor FRα using loop dynamics of three essential loops present in the protein along with polar stabilization interactions provided by Asp55 and Glu86 residues present in the active site. The f-CNO effectively decreased cancer cell viability with a low IC50 value (the concentration that led to 50% killing of the cells). The cell-based Franz diffusion assay was performed to study the drug release profile. The f-CNO-BCNUs also decreased the mitochondrial membrane potential of U87 cells, increased reactive oxygen species release, and caused a loss of mitochondrial membrane integrity. The f-CNOs also increased the percentage of apoptotic cells observed by the Annexin V assay. Based on observed results, it can be concluded that the f-CNO-BCNU efficiently targets the cancer cells, enhances the bioavailability of carmustine, and can be used as a smart chemotherapeutic agent. This strategy offers better patient compliance and greater bioavailability of the drug.

The coordinated action of the enzymes in the L-lysine biosynthetic pathway and how to inhibit it for antibiotic targets.

BACKGROUND: Antimicrobial resistance is a global health issue that requires immediate attention in terms of new antibiotics and new antibiotic targets. The l-lysine biosynthesis pathway (LBP) is a promising avenue for drug discovery as it is essential for bacterial growth and survival and is not required by human beings. SCOPE OF REVIEW: The LBP involves a coordinated action of fourteen different enzymes distributed over four distinct sub-pathways. The enzymes involved in this pathway belong to different classes, such as aspartokinase, dehydrogenase, aminotransferase, epimerase, etc. This review provides a comprehensive account of the secondary and tertiary structure, conformational dynamics, active site architecture, mechanism of catalytic action, and inhibitors of all enzymes involved in LBP of different bacterial species. MAJOR CONCLUSIONS: LBP offers a wide scope for novel antibiotic targets. The enzymology of a majority of the LBP enzymes is well understood, although these enzymes are less widely studied in the critical pathogens (according to the 2017 WHO report) that require immediate attention. In particular, the enzymes in the acetylase pathway, DapAT, DapDH, and Aspartokinase in critical pathogens have received little attention. High throughput screening for inhibitor design against the enzymes of lysine biosynthetic pathway is rather limited, both in number and in the extent of success. GENERAL SIGNIFICANCE: This review can serve as a guide for the enzymology of LBP and help in identifying new drug targets and designing potential inhibitors.