Atomic Insight into the Enzymatic Selectivity of Acetyltransferase for Endogenous Polyamines.

The N1-Spermidine/spermine acetyltransferase (SSAT) serves as the rate-limiting enzyme in the polyamine metabolism pathway, specifically catalyzing the acetylation of spermidine, spermine, and other specific polyamines. The source of its enzymatic selectivity remains elusive. Here, we used quantum mechanics and molecular mechanics simulations combined with various technologies to explore the enzymatic mechanism of SSAT for endogenous polyamines from an atomic perspective. The static binding and chemical transformation were considered. The binding affinity was identified to be dependent on the protonated state of polyamine. The order of the binding affinity for Spm, Spd, and Put is consistent with the experimental results, which is also verified by the dynamic separation of polyamine and SSAT. Hydrogen bond interactions and salt bridges contribute most, and the common hot residues were identified. In addition, the transfer of acetyl and proton between polyamine and AcCoA was discovered to follow a concerted mechanism, and thermodynamic properties are responsible for the catalytic efficiency of SSAT. This work may be helpful for the development of polyamine derivatives based on catalysis to regulate polyamine metabolism.

Enzymatic-related network of catalysis, polyamine, and tumors for acetylpolyamine oxidase: from calculation to experiment.

The regulation of enzymes and development of polyamine analogs capable of controlling the dynamics of endogenous polyamines to achieve anti-tumor effects is one of the biggest challenges in polyamine research. However, the root of the problem remains unsolved. This study represents a significant milestone as it unveils, for the first time, the comprehensive catalytic map of acetylpolyamine oxidase that includes chemical transformation and product release kinetics, by utilizing multiscale simulations with over six million dynamical snapshots. The transportation of acetylspermine is strongly exothermic, and high binding affinity of enzyme and reactant is observed. The transfer of hydride from polyamine to FAD is the rate-limiting step, via an H-shift coupled electron transfer mechanism. The two products are released in a detour stepwise mechanism, which also impacts the enzymatic efficiency. Inspired by these mechanistic insights into enzymatic catalysis, we propose a novel strategy that regulates the polyamine level and catalytic progress through the action of His64. Directly suppressing APAO by mutating His64 further inhibited growth and migration of tumor cells and tumor tissue in vitro and in vivo. Therefore, the network connecting microcosmic and macroscopic scales opens up new avenues for designing polyamine compounds and conducting anti-tumor research in the future.

Cryo-EM structure and molecular dynamic simulations explain the enhanced stability and ATP activity of the pathological chaperonin mutant.

Chaperonins Hsp60s are required for cellular vitality by assisting protein folding in an ATP-dependent mechanism. Although conserved, the human mitochondrial mHsp60 exhibits molecular characteristics distinct from the E. coli GroEL, with different conformational assembly and higher subunit association dynamics, suggesting a different mechanism. We previously found that the pathological mutant mHsp60V72I exhibits enhanced subunit association stability and ATPase activity. To provide structural explanations for the V72I mutational effects, here we determined a cryo-EM structure of mHsp60V72I. Our structural analysis combined with molecular dynamic simulations showed mHsp60V72I with increased inter-subunit interface, binding free energy, and dissociation force, all contributing to its enhanced subunit association stability. The gate to the nucleotide-binding (NB) site in mHsp60V72I mimicked the open conformation in the nucleotide-bound state with an additional open channel leading to the NB site, both promoting the mutant's ATPase activity. Our studies highlight the importance of mHsp60's characteristics in its biological function.

Static Binding and Dynamic Transporting-Based Design of Specific Ring-Chain-Ring Acetylcholinesterase Inhibitor: From Galantamine to Natural Product.

Acetylcholinesterase (AChE) is a key target for the current symptomatic treatment of Alzheimer's disease, and galantamine is a clinical anticholinesterase drug with transiently acting characteristic and good selectivity for AChE. The present theoretical-experimental work improves the drug's residence time without reducing the inhibition effect, thus providing a crucial breakthrough for modifying the inhibitor of AChE with better kinetic behavior. The static binding and dynamic delivery properties acquired from atomic view reveal that the galantamine simply occupies a catalytic anionic site, and its release from AChE needs only ∼8.6 kcal/mol. Both of these may cause the short residence time of galantamine. The hotspots and most favorable transport mechanism are identified, and the hydrogen bond and aromatic stacking interactions are observed to play crucial roles for galantamine binding and release in AChE. The typical peripheral anionic site arisen at the delivery process would provide another key occupation to enhance the anti-release ability for inhibitors. The compound with "specific-ring-chain-ring" framework with detailed beneficial modification scheme is summarized, which may improve the residence time of the inhibitor in AChE. The thermodynamic and dynamic properties of galantamine derivatives are also studied. Based on dictamnine, a natural alkaloid, two novel eligible derivatives are designed, synthesized and evaluated, which verifies our prediction. Multiple computational approaches and experimental combinations probably provide a train of thought from both static and dynamic views to modify or design appropriate inhibitors on the basis of specific binding and transportation features.

Combination of multiple methods and views for recognition, transportation, and structure-guided modification of lysine-specific demethylase phenylcyclopropylamine inhibitor.

Lysine-Specific Demethylase 1 (LSD1) is a typical histone-specific demethylase, which plays an important role in protein methylation modification. It is a member of the amine oxidase family (MAO) that specifically removes methyl groups from monomethylated H3K4, dimethylated H3K4 and H3K9 sites associated with tumorigenesis. Phenylcyclopropylamine derivatives are a class of specific LSD1 inhibitors, drawing attention due to their high efficiency. Here, extensive molecular dynamics (MD) simulations are combined with a three-dimensional quantitative structure-activity relationship (3D-QSAR) in order to design a new phenylcyclopropylamine inhibitor from multiple perspectives. In a ligand-oriented point of view, a 3D-QSAR model with comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) can be built based on the 55 phenylcyclopropylamine compounds targeting LSD1 obtained experimentally. The aromatic and piperazine rings are identified as the potential key groups regulating the activity of the compounds. In an interaction-oriented view, the representative compound is defined with the highest inhibitory efficiency. The binding and delivery mechanism and conformational dependence of activity, including channel and dynamic properties, are studied using RAMD and umbrella sampling technologies. The direct hydrogen bond and conjugated interactions are identified as a major driving force in this procedure. The dominant region of the phenylcyclopropylamine influences the free energy and detects the key residues in recognition and delivery. On the basis of both the ligand and interaction, a series of new inhibitor structures were designed, and two of them showed better efficiency. In order to select the inhibitor with a longer residence time, a comparison is conducted between the designed inhibitors and the experimentally obtained inhibitor from the perspective of static binding and dynamic delivery properties. This work creates new guidance for the phenylcyclopropylamine inhibitor design of LDS1 by combining the ligand and receptor, considering both static and dynamic properties. This scheme could be applied in other inhibitor design systems.