Structural and Functional Characterization of Camelus dromedarius Glutathione Transferase M1-1

Glutathione transferases (GSTs; EC. 2.5.1.18) are a large family of multifunctional enzymes that play crucial roles in the metabolism and inactivation of a broad range of xenobiotic compounds. In the present work, we report the kinetic and structural characterization of the isoenzyme GSTM1-1 from Camelus dromedarius (CdGSTM1-1). The CdGSΤM1-1 was expressed in E. coli BL21 (DE3) and was purified by affinity chromatography. Kinetics analysis showed that the enzyme displays a relative narrow substrate specificity and restricted ability to bind xenobiotic compounds. The crystal structures of CdGSΤM1-1 were determined by X-ray crystallography in complex with the substrate (GSH) or the reaction product (S-p-nitrobenzyl-GSH), providing snapshots of the induced-fit catalytic mechanism. The thermodynamic stability of CdGSTM1-1 was investigated using differential scanning fluorimetry (DSF) in the absence and in presence of GSH and S-p-nitrobenzyl-GSH and revealed that the enzyme’s structure is significantly stabilized by its ligands. The results of the present study advance the understanding of camelid GST detoxification mechanisms and their contribution to abiotic stress adaptation in harsh desert conditions.

Noble-Metal-Based Catalytic Oxidation Technology Trends for Volatile Organic Compound (VOC) Removal

Volatile organic compounds (VOCs) are toxic and are considered the most important sources for the formation of photochemical smog, secondary organic aerosols (SOAs), and ozone. These can also greatly affect the environment and human health. For this reason, VOCs are removed by applying various technologies or reused after recovery. Catalytic oxidation for VOCs removal is widely applied in the industry and is regarded as an efficient and economical method compared to other VOCs removal technologies. Currently, a large amount of VOCs are generated in industries with solvent-based processes, and the ratio of aromatic compounds is high. This paper covers recent catalytic developments in VOC combustion over noble-metal-based catalysts. In addition, this report introduces recent trends in the development of the catalytic mechanisms of VOC combustion and the deactivation of catalysts, such as coke formation, poisoning, sintering, and catalyst regeneration. Since VOC oxidation by noble metal catalysts depends on the support of and mixing catalysts, an appropriate catalyst should be used according to reaction characteristics. Moreover, noble metal catalysts are used together with non-noble metals and play a role in the activity of other catalysts. Therefore, further elucidation of their function and catalytic mechanism in VOC removal is required.

Structural Aspects of LIMK Regulation and Pharmacology

Malfunction of the actin cytoskeleton is linked to numerous human diseases including neurological disorders and cancer. LIMK1 (LIM domain kinase 1) and its paralogue LIMK2 are two closely related kinases that control actin cytoskeleton dynamics. Consequently, they are potential therapeutic targets for the treatment of such diseases. In the present review, we describe the LIMK conformational space and its dependence on ligand binding. Furthermore, we explain the unique catalytic mechanism of the kinase, shedding light on substrate recognition and how LIMK activity is regulated. The structural features are evaluated for implications on the drug discovery process. Finally, potential future directions for targeting LIMKs pharmacologically, also beyond just inhibiting the kinase domain, are discussed.

Catalytically active holo Homo sapiens adenosine deaminase I adopts a closed conformation

Homo sapiens adenosine deaminase 1 (HsADA1; UniProt P00813) is an immunologically relevant enzyme with roles in T-cell activation and modulation of adenosine metabolism and signaling. Patients with genetic deficiency in HsADA1 suffer from severe combined immunodeficiency, and HsADA1 is a therapeutic target in hairy cell leukemias. Historically, insights into the catalytic mechanism and the structural attributes of HsADA1 have been derived from studies of its homologs from Bos taurus (BtADA) and Mus musculus (MmADA). Here, the structure of holo HsADA1 is presented, as well as biochemical characterization that confirms its high activity and shows that it is active across a broad pH range. Structurally, holo HsADA1 adopts a closed conformation distinct from the open conformation of holo BtADA. Comparison of holo HsADA1 and MmADA reveals that MmADA also adopts a closed conformation. These findings challenge previous assumptions gleaned from BtADA regarding the conformation of HsADA1 that may be relevant to its immunological interactions, particularly its ability to bind adenosine receptors. From a broader perspective, the structural analysis of HsADA1 presents a cautionary tale for reliance on homologs to make structural inferences relevant to applications such as protein engineering or drug development.

Mechanistic analysis of carbon–carbon bond formation by deoxypodophyllotoxin synthase

Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate–dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS’s catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel–Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.

Exploring the Catalytic Mechanism of the RNA Cap Modification by nsp16-nsp10 Complex of SARS-CoV-2 through a QM/MM Approach

The inhibition of key enzymes that may contain the viral replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have assumed central importance in drug discovery projects. Nonstructural proteins (nsps) are essential for RNA capping and coronavirus replication since it protects the virus from host innate immune restriction. In particular, nonstructural protein 16 (nsp16) in complex with nsp10 is a Cap-0 binding enzyme. The heterodimer formed by nsp16-nsp10 methylates the 5′-end of virally encoded mRNAs to mimic cellular mRNAs and thus it is one of the enzymes that is a potential target for antiviral therapy. In this study, we have evaluated the mechanism of the 2′-O methylation of the viral mRNA cap using hybrid quantum mechanics/molecular mechanics (QM/MM) approach. It was found that the calculated free energy barriers obtained at M062X/6-31+G(d,p) is in agreement with experimental observations. Overall, we provide a detailed molecular analysis of the catalytic mechanism involving the 2′-O methylation of the viral mRNA cap and, as expected, the results demonstrate that the TS stabilization is critical for the catalysis.

Enzyme Inhibitors: The Best Strategy to Tackle Superbug NDM-1 and Its Variants

Multidrug bacterial resistance endangers clinically effective antimicrobial therapy and continues to cause major public health problems, which have been upgraded to unprecedented levels in recent years, worldwide. β-Lactam antibiotics have become an important weapon to fight against pathogen infections due to their broad spectrum. Unfortunately, the emergence of antibiotic resistance genes (ARGs) has severely astricted the application of β-lactam antibiotics. Of these, New Delhi metallo-β-lactamase-1 (NDM-1) represents the most disturbing development due to its substrate promiscuity, the appearance of variants, and transferability. Given the clinical correlation of β-lactam antibiotics and NDM-1-mediated resistance, the discovery, and development of combination drugs, including NDM-1 inhibitors, for NDM-1 bacterial infections, seems particularly attractive and urgent. This review summarizes the research related to the development and optimization of effective NDM-1 inhibitors. The detailed generalization of crystal structure, enzyme activity center and catalytic mechanism, variants and global distribution, mechanism of action of existing inhibitors, and the development of scaffolds provides a reference for finding potential clinically effective NDM-1 inhibitors against drug-resistant bacteria.

Design of a graphene oxide-BODIPY conjugate for glutathione depletion and photodynamic therapy

Boron dipyrromethene derivates (BODIPYs) are promising photosensitisers (PSs) for cancer treatment using photodynamic therapy (PDT). This study investigates the functionalisation of graphene oxide (GO) with a BODIPY derivate for glutathione (GSH) depletion and PDT. The functionalisation of GO with a 3,5-dichloro-8-(4-boronophenyl) BODIPY via a diol derivatisation with the phenyl boronic acid moiety at the meso position of the BODIPY core, allowed to preserve the intrinsic properties of GO. We demonstrated that both chlorine atoms were substituted by GSH in the presence of glutathione transferase (GST), inducing a relevant bathochromic shift in the absorption/emission features and thus generating the active PS. Ex vitro assessment using cell lysates containing cytoplasmatic GST revealed the intracellular catalytic mechanism for the nucleophilic substitution of the GO-BODIPY adduct with GSH. Confocal microscopy studies showed important differences in the cellular uptake of free BODIPY and GO-BODIPY and revealed the coexistence of GO-BODIPY, GO-BODIPY-GS, and GO-BODIPY-GS2 species inside vesicles and in the cytoplasm of the cells after 24 h of incubation. In vitro biocompatibility and safety of GO and GO-BODIPY were evaluated in 2D and 3D models of prostate adenocarcinoma cells (PC-3), where no toxicity was observed up to 100 µg/mL of GO/GO-BODIPY in all treated groups 24 h post-treatment (cell viability > 90%). Only a slight decrease to 80% at 100 µg/mL was observed after 48 h of incubation. We demonstrated the efficacy of a GO adduct containing an α-chlorine-substituted BODIPY for the simultaneous depletion of intracellular GSH and the photogeneration of reactive oxygen species using a halogen white light source (5.4 mW/cm2) with a maximum in the range of 500-800 nm, which significantly reduced cell viability (< 50%) after irradiation. Our study provides a new vision on how to apply BODIPY derivates and potentiate the toxicity of PDT in prostate and other types of cancer.