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β-lactamase production with vast catalytic divergence in the pathogenic strain limits the antibiotic spectrum in the clinical environment. Class A carbapenemase shares significant sequence similarities, structural features, and common catalytic mechanisms although their resistance spectrum differs from class A β-lactamase in carbapenem and monobactam hydrolysis. In other words, it limited the antibiotic treatment option against infection, causing carbapenemase-producing superbugs. Ftu-1 is a class A β-lactamase expressed by the Francisella tularensisstrain, a potent causative organism of tularemia. The chromosomally encoded class A β-lactamase shares two conserved cysteine residues, a common characteristic of a carbapenemase, and a distinctive class in the phylogenetic tree. Complete biochemical and biophysical characterization of the enzyme was performed to understand the overall stability and environmental requirements to perform optimally. To comprehend the enzyme–drug interaction and its profile toward various chemistries of β-lactam and β-lactamase inhibitors, comprehensive kinetic and thermodynamic analyses were conducted using various β-lactam drugs. The dynamic property of Ftu-1 β-lactamase was also predicted using molecular dynamics (MD) simulation to compare its loop flexibility and ligand binding with other related class A β-lactamases. Overall, this study fosters a comprehensive understanding of Ftu-1, proposed to be an intermediate class by characterizing its kinetic profiling, stability by biochemical and biophysical methodologies, and susceptibility profiling. This understanding would be beneficial for the design of new-generation therapeutics.

AbstractThe innate immune system has an important role in developing the initial resistance to virus infection, and the ability of oligoadenylate synthetase to overcome viral evasion and enhance innate immunity is already established in humans. In the present study, we have tried to explore the molecular and structural variations present in Sahiwal (indigenous) and crossbred (Frieswal) cattle to identify the molecular mechanism of action of OAS1 gene in activation of innate immune response. The significant changes in structural alignment in terms of orientation of loops, shortening of β-sheets and formation of 3–10 α-helix was noticed in Sahiwal and Frieswal cattle. Further, it has been observed that OAS1 from Sahiwal had better binding with APC and DTP ligand than Frieswal OAS1. A remarkable change was seen in orientation at the nucleoside base region of both the ligands, which are bound with OAS1 protein from Frieswal and Sahiwal cattle. The Molecular Dynamic study of apo and ligand complex structures was provided more insight towards the stability of OAS1 from both cattle. This analysis displayed that the Sahiwal cattle protein has more steady nature throughout the simulation and has better binding towards Frieswal in terms of APC and DTP binding. Thus, OAS1 protein is the potential target for explaining the innate immune response in Sahiwal than Frieswal.Communicated by Ramaswamy H. Sarma

Angiotensin converting enzyme-I (ACE-I) is a key therapeutic target of the renin–angiotensin–aldosterone system (RAAS), the central pathway of blood pressure regulation. Food-derived peptides with ACE-I inhibitory activities are receiving significant research attention. However, identification of ACE-I inhibitory peptides from different food proteins is a labor-intensive, lengthy, and expensive process. For successful identification of potential ACE-I inhibitory peptides from food sources, a machine learning and structural bioinformatics-based web server has been developed and reported in this study. The web server can take input in the FASTA format or through UniProt ID to perform the in silicogastrointestinal digestion and then screen the resulting peptides for ACE-I inhibitory activity. This unique platform provides elaborated structural and functional features of the active peptides and their interaction with ACE-I. Thus, it can potentially enhance the efficacy and reduce the time and cost in identifying and characterizing novel ACE-I inhibitory peptides from food proteins. URL: http://hazralab.iitr.ac.in/ahpp/index.php.

AbstractFunctional foods are emerging as essential healthy nutritional component due to their abundant wellbeing benefits. Especially the food-derived peptides are considered as key components for playing their biologically active roles. One such robust therapeutics that already exploited with food peptides that help treating high blood pressure via targeting Angiotensin-Converting Enzyme (ACE). This in silicostudy demonstrated the inhibitory potential of antihypertensive peptides derived from food sources. This study involves an intensive structure-based analysis of enzyme-peptide interactions using Molecular Dynamics (MD) simulations. Interestingly, this study will help us to get deeper understanding on how food peptides achieve successful inhibition of ACE. In this study, the peptide-enzyme complexes revealed two binding pockets, A and B, on either side of the active site Zn atom. Pocket B has a smaller binding site volume than pocket A, comprised of β-sheets and the active site opening cleft. The interface of the binding sites showed that the enzyme structure was negative to neutral charge, and the peptide structure was positive to neutral charge. The dynamics of complex structures of seven highly potential peptides were performed for 20 ns each at 300 K. Comparative analysis of RMSD, RMSF and binding energies show the enzyme-peptide complexes and the overall stability of apo-enzyme. Importantly, two peptides AFKAWAVAR and IWHHTF showed the highest variation in their RMSD as compared to the apo-enzyme. This study will further be useful for the assessment of the characteristics to predict novel inhibitory peptides that can be generated from food proteins.Communicated by Ramaswamy H. Sarma

The low toxicity of acyclovir (ACV) is mainly due to the fact that human nucleoside kinases have undetectable phosphorylation rates with this acyclic guanine analogue. In contrast, herpes virus thymidine kinase (HSV1-TK) readily activates ACV. We wanted to understand why human deoxycytidine kinase (dCK), which is related to HSV1-TK and phosphorylates deoxyguanosine, does not accept acyclic guanine analogues as substrates. Therefore, we crystallized dCK in complex with ACV at the nucleoside phosphoryl acceptor site and UDP at the phosphoryl donor site. The structure reveals that while ACV does bind at the dCK active site, it does so adopting a nonproductive conformation. Despite binding ACV, the enzyme remains in the open, inactive state. In comparison to ACV binding to HSV1-TK, in dCK, the nucleoside base adopts a different orientation related by about a 60° rotation. Our analysis suggests that dCK would phosphorylate acyclic guanine analogues if they can induce a similar rotation.

Kluyveromyces marxianusIIPE453 can utilize biomass-derived fermentable sugars for xylitol and ethanol fermentation. In this study, the xylitol production in the native strain was improved by overexpression of endogenous d-xylose reductase gene. A suitable expression cassette harboring the gene of interest was constructed and incorporated in the native yeast. qPCR analysis demonstrated the 2.1-fold enhancement in d-xylose reductase transcript levels in the modified strain with 1.62-fold enhancement in overall xylitol yield without affecting its ethanol fermenting capacity. Material balance analysis on 2 kg of sugar cane bagasse-derived fermentable sugars illustrated an excess of 58.62 ± 0.15 g of xylitol production by transformed strain in comparison to the wild variety with similar ethanol yield. The modified strain can be suitably used as a single biocatalyst for multiproduct biorefinery application.

Comparative molecular dynamics simulations of chemotaxis protein “CheY” from thermophilic origin Thermotoga maritimaand its mesophilic counterpart Salmonella entericahave been performed for 10 ns each at 300 and 350 K, and 20 ns each at 400 and 450 K. The trajectories were analyzed in terms of different factors like root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessible surface area, H-bonds, salt bridge content, and protein–solvent interactions which indicate distinct differences between the two of them. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their optimum growth temperatures. However, at higher temperatures mesophilic protein shows increased overall flexibility than its thermophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are nonoverlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar direction of motions are observed for both proteins at 300 K. Overall, the mesophilic protein leads to increased conformational sampling of the phase space than its thermophilic counterpart. This is the first ever study of thermostability of CheY protein homologs by using protein dynamism as a main impact. Our study might be used as a model for studying the molecular basis of thermostability of two homologous proteins from two organisms living at different temperatures with less visible differences.

ABSTRACTThe current emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for novel treatment strategies. Recently, BlaC, the principal β-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. The combination of meropenem and clavulanic acid, which inhibits BlaC, was found to be effective against even extensively drug-resistant M. tuberculosisstrains when tested in vitro. Yet there is significant concern that drug resistance against this combination will also emerge. To investigate the potential of BlaC to evolve variants resistant to clavulanic acid, we introduced substitutions at important amino acid residues of M. tuberculosisBlaC (R220, A244, S130, and T237). Whereas the substitutions clearly led to in vitroclavulanic acid resistance in enzymatic assays but at the expense of catalytic activity, transformation of variant BlaCs into an M. tuberculosisH37Rv background revealed that impaired inhibition of BlaC did not affect inhibition of growth in the presence of ampicillin and clavulanate. From these data we propose that resistance to β-lactam–β-lactamase inhibitor combinations will likely not arise from structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be part of a successful treatment regimen against M. tuberculosis.

Tuberculosis is a common, and in many cases lethal, infectious disease caused by various strains of Mycobacterium, usually Mycobacterium tuberculosis. (Kumar et al., 2007) In addition, co-infection with Mycobacterium tuberculosisand HIV (TB/HIV), especially in Africa, and multidrug resistant and extensively drug-resistant tuberculosis in all regions, (WHO, 2010) makes it important to develop novel therapeutics against this bacterium. Penicillin like β-Lactam antibiotics are among the most clinically prescribed drugs for anti-bacterial therapeutics. The general mechanism of action involved the inhibition of enzyme d,d-transpeptidases, which takes part in the biosynthesis of the bacterial cell wall (Heesemann, 1993). A major strategy of bacterial resistance to β-lactams is the production of β-lactamases that catalyze the hydrolysis of β-lactams, leading to the inactivation of the antibiotics. β-Lactams have not been used in clinical practice to treat TB infections, because an active penicillinase was reported in M. tuberculosis(Lessel, 1996). BlaC is a class A β-lactamase that contains a nucleophilic serine residue (Ser70) and shares sequence homology with the penicillin-binding protein domain of the ancestral d,d-transpeptidases. Recent studies including our lab show that β-lactam drugs like Clavulanate, Carbapenem, and Meropenem are used primarily against this type of resistant bacteria (Hugonnet et al., 2009). β-lactamase induces the same acetylating reaction with all of these drugs but cannot induce deacetylation. As a result, those drugs remain attached with β-lactamase even after the distortion of their β-lactam ring. At this time, secondary treatment has been done by applying previously used potent penicillin like β-lactam drugs with this primarily treated β-lactamase. In current study, we conducted kinetic and mass spectrometric analysis of different BlaC inhibitors, like NXL104 (Xu et al., 2012) and showed that how they quantitatively inactivates BlaC by forming a carbamyl linkage with the enzyme. In addition, we determined the three-dimensional structures of the different reactive forms of these drugs for better understanding the undergoing mechanisms involved in this inhibition process. Based on our understanding, we are trying to develop novel small molecules with better inhibitory process.