Structural and Evolutionary Analyses of PR-4 SUGARWINs Points to a Different Pattern of Protein Function.

SUGARWINs are PR-4 proteins associated with sugarcane defense against phytopathogens. Their expression is induced in response to damage by Diatraea saccharalis larvae. These proteins play an important role in plant defense, in particular against fungal pathogens, such as Colletothricum falcatum (Went) and Fusarium verticillioides. The pathogenesis-related protein-4 (PR-4) family is a group of proteins equipped with a BARWIN domain, which may be associated with a chitin-binding domain also known as the hevein-like domain. Several PR-4 proteins exhibit both chitinase and RNase activity, with the latter being associated with the presence of two histidine residues H11 and H113 (BARWIN) [H44 and H146, SUGARWINs] in the BARWIN-like domain. In sugarcane, similar to other PR-4 proteins, SUGARWIN1 exhibits ribonuclease, chitosanase and chitinase activities, whereas SUGARWIN2 only exhibits chitosanase activity. In order to decipher the structural determinants involved in this diverse range of enzyme specificities, we determined the 3-D structure of SUGARWIN2, at 1.55Å by X-ray diffraction. This is the first structure of a PR-4 protein where the first histidine has been replaced by asparagine and was subsequently used to build a homology model for SUGARWIN1. Molecular dynamics simulations of both proteins revealed the presence of a flexible loop only in SUGARWIN1 and we postulate that this, together with the presence of the catalytic histidine at position 42, renders it competent as a ribonuclease. The more electropositive surface potential of SUGARWIN1 would also be expected to favor complex formation with RNA. A phylogenetic analysis of PR-4 proteins obtained from 106 Embryophyta genomes showed that both catalytic histidines are widespread among them with few replacements in these amino acid positions during the gene family evolutionary history. We observe that the H11 replacement by N11 is also present in two other sugarcane PR-4 proteins: SUGARWIN3 and SUGARWIN4. We propose that RNase activity was present in the first Embryophyta PR-4 proteins but was recently lost in members of this family during the course of evolution.

SARS-CoV-2 mutations in Brazil: from genomics to putative clinical conditions.

Due to the high rate of transmissibility, Brazil became the new COVID-19 outbreak epicenter and, since then, is being monitored to understand how SARS-CoV-2 mutates and spreads. We combined genomic and structural analysis to evaluate genomes isolated from different regions of Brazil and show that the most prevalent mutations were located in the S, N, ORF3a and ORF6 genes, which are involved in different stages of viral life cycle and its interaction with the host cells. Structural analysis brought to light the positions of these mutations on protein structures, contributing towards studies of selective structure-based drug discovery and vaccine development.

Oleuropein Protects Against Cerebral Ischemia Injury in Rats: Molecular Docking, Biochemical and Histological Findings.

This study was designed to evaluate the underlying protective mechanisms of oleuropein involved in alleviating brain damage in a rat model of ischemic stroke. Male Wistar rats were divided into four groups; Control, stroke (MCAO), MCAO + clopidogrel (Clop) and MCAO + oleuropein (Ole). Results showed that the MCAO group evidenced significant brain edema (+ 9%) as well as increases of plasma cardiac markers such as lactate deshydrogenase (LDH), creatine kinase (CK-MB), fibrinogen and Trop-T by 11 %, 43%, 168 and 590%, respectively, as compared to the control group. Moreover, infarcted rats exhibited remarkable elevated levels of angiotensin converting enzyme (ACE), both in plasma and brain tissue, with astrocyte swelling and necrotic neurons in the infarct zone, hyponatremia, and increased rate of thiobarbituric acid-reactive substances (TBARS) by 89% associated with decreases in the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (Cat) by 51%, 44 and 42%, respectively, compared to normal control rats. However, MCAO rats treated with oleuropein underwent mitigation of cerebral edema, correction of hyponatremia, remarkable decrease of plasma fibrinogen and cardiac dysfunctional enzymes, inhibition of ACE activity and improvement of oxidative stress status in brain tissue. Furthermore, in silico analysis showed considerable inhibitions of ACE, protein disulfide isomerase (PDI) and TGF-ß1, an indicative of potent anti-embolic properties. Overall, oleuropein offers a neuroprotective effect against ischemic stroke through its antioxidative and antithrombotic activities.

Theoretical characterization of the shikimate 5-dehydrogenase reaction from Mycobacterium tuberculosis by hybrid QC/MM simulations and quantum chemical descriptors.

In this study, we have investigated the enzyme shikimate 5-dehydrogenase from the causative agent of tuberculosis, Mycobacterium tuberculosis. We have employed a mixture of computational techniques, including molecular dynamics, hybrid quantum chemical/molecular mechanical potentials, relaxed surface scans, quantum chemical descriptors and free-energy simulations, to elucidate the enzyme's reaction pathway. Overall, we find a two-step mechanism, with a single transition state, that proceeds by an energetically uphill hydride transfer, followed by an energetically downhill proton transfer. Our mechanism and calculated free energy barrier for the reaction, 64.9 kJ mol- 1, are in good agreement with those predicted from experiment. An analysis of quantum chemical descriptors along the reaction pathway indicated a possibly important, yet currently unreported, role of the active site threonine residue, Thr65.

Elucidating Enzymatic Catalysis Using Fast Quantum Chemical Descriptors.

In general, computational simulations of enzymatic catalysis processes are thermodynamic and structural surveys to complement experimental studies, requiring high level computational methods to match accurate energy values. In the present work, we propose the usage of reactivity descriptors, theoretical quantities calculated from the electronic structure, to characterize enzymatic catalysis outlining its reaction profile using low-level computational methods, such as semiempirical Hamiltonians. We simulate three enzymatic reactions paths, one containing two reaction coordinates and without prior computational study performed, and calculate the reactivity descriptors for all obtained structures. We observed that the active site local hardness does not change substantially, even more so for the amino-acid residues that are said to stabilize the reaction structures. This corroborates with the theory that activation energy lowering is caused by the electrostatic environment of the active sites. Also, for the quantities describing the atom electrophilicity and nucleophilicity, we observed abrupt changes along the reaction coordinates, which also shows the enzyme participation as a reactant in the catalyzed reaction. We expect that such electronic structure analysis allows the expedient proposition and/or prediction of new mechanisms, providing chemical characterization of the enzyme active sites, thus hastening the process of transforming the resolved protein three-dimensional structures in catalytic information.

Piperazine derivatives: synthesis, inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase and SAR studies.

The Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA) catalyzes hydride transfer to long-chain enoyl thioester substrates. MtInhA is a member of the mycobacterial type II dissociated fatty acid biosynthesis system, and is the bona fide target for isoniazid, the most prescribed drug for tuberculosis treatment. Here, a series of piperazine derivatives was synthesized and screened as MtInhA inhibitors, which resulted in the identification of compounds with IC50 values in the submicromolar range. A structure-activity relationship (SAR) evaluation indicated the importance of the chemical environment surrounding the carbonyl group for inhibition. In addition, the structure of one selected compound was supported by crystallographic studies, and experimental geometrical values were compared with semi-empirical quantum chemical calculations. Furthermore, the mode of inhibition and inhibitory dissociation constants were determined for the nine most active compounds. These findings suggest that these 9H-fluoren-9-yl-piperazine-containing compounds interact with MtInhA at the enoyl thioester (2-trans-dodecenoyl-CoA) substrate binding site.

Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity.

Superoxide dismutases (SODs) are a crucial class of enzymes in the combat against intracellular free radical damage. They eliminate superoxide radicals by converting them into hydrogen peroxide and oxygen. In spite of their very different life cycles and infection strategies, the human parasites Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei are known to be sensitive to oxidative stress. Thus the parasite Fe-SODs have become attractive targets for novel drug development. Here we report the crystal structures of FeSODs from the trypanosomes T. brucei at 2.0 A and T. cruzi at 1.9 A resolution, and that from P. falciparum at a higher resolution (2.0 A) to that previously reported. The homodimeric enzymes are compared to the related human MnSOD with particular attention to structural aspects which are relevant for drug design. Although the structures possess a very similar overall fold, differences between the enzymes at the entrance to the channel which leads to the active site could be identified. These lead to a slightly broader and more positively charged cavity in the parasite enzymes. Furthermore, a statistical coupling analysis (SCA) for the whole Fe/MnSOD family reveals different patterns of residue coupling for Mn and Fe SODs, as well as for the dimeric and tetrameric states. In both cases, the statistically coupled residues lie adjacent to the conserved core surrounding the metal center and may be expected to be responsible for its fine tuning, leading to metal ion specificity.