Biochemical Properties and Roles of DprA Protein in Bacterial Natural Transformation, Virulence, and Pilin Variation.

Natural transformation enables bacteria to acquire DNA from the environment and contributes to genetic diversity, DNA repair, and nutritional requirements. DNA processing protein A (DprA) receives incoming single-stranded DNA and assists RecA loading for homology-directed natural chromosomal transformation and DNA strand annealing during plasmid transformation. The dprA gene occurs in the genomes of all known bacteria, irrespective of their natural transformation status. The DprA protein has been characterized by its molecular, cellular, biochemical, and biophysical properties in several bacteria. This review summarizes different aspects of DprA biology, collectively describing its biochemical properties, molecular interaction with DNA, and function interaction with bacterial RecA during natural transformation. Furthermore, the roles of DprA in natural transformation, bacterial virulence, and pilin variation are discussed.

Insights into the evolution of mutations in SARS-CoV-2 non-spike proteins.

The COVID-19 pandemic has been driven by the emergence of SARS-CoV-2 variants with mutations across all the viral proteins. Although mutations in the spike protein have received significant attention, understanding the prevalence and potential impact of mutations in other viral proteins is essential for comprehending the evolution of SARS-CoV-2. Here, we conducted a comprehensive analysis of approximately 14 million sequences of SARS-CoV-2 deposited in the GISAID database until December 2022 to identify prevalent mutations in the non-spike proteins at the global and country levels. Additionally, we evaluated the energetics of each mutation to better understand their impact on protein stability. While the consequences of many mutations remain unclear, we discuss potential structural and functional significance of some mutations. Our study highlights the ongoing evolutionary process of SARS-CoV-2 and underscores the importance of understanding changes in non-spike proteins.

Mechanistic and evolutionary insights into alkaline phosphatase superfamily through structure-function studies on Sphingomonas alkaline phosphatase.

Alkaline phosphatases (APs), represented by E. coli AP (ECAP), employ an arginine residue to stabilize the phosphoryl group in the active site; whereas, AP from Sphingomonas (SPAP) shows a unique combination of substrate-binding residues; Thr89, Asn110, Lys171, and Arg173. Although such combination has been observed only in SPAP, these residues are present separately in different members of the AP superfamily. Here, we establish the presence of two distinct classes of APs; ECAP-type and SPAP-type. Bioinformatic analyses show that SPAP-type of APs are widely distributed in the bacterial kingdom. The role of active site residues in the catalytic mechanism has been delineated through a set of crystal structures reported here. These structures, representing different stages of the reaction pathway provide wealth of information for the catalytic mechanism. Despite critical differences in the substrate binding residues, SPAP follows a mechanism similar to that of ECAP-type of APs. Structure-based phylogenetic analysis suggests that SPAP and ECAP may have diverged very early during the evolution from a common ancestor. Moreover, it is proposed that the SPAP-type of APs are fundamental members of the AP superfamily and are more closely related to other members of the superfamily as compared to the ECAP-type of APs.

Diselenide-derivative of 3-pyridinol targets redox enzymes leading to cell cycle deregulation and apoptosis in A549 cells.

The aim of present study was to understand the mechanism of action of 2,2'-diselenobis(3-pyridinol) or DISPOL in human lung cancer (A549) cells. A549 cells were treated with 10 µM (∼IC50) of DISPOL for varying time points to corelate the intracellular redox changes with its cytotoxic effect. The results indicated that DISPOL treatment led to a time dependant decrease in the basal level of reactive oxygen species (ROS). Additionally, DISPOL treatment elevated the ratio of reduced (GSH) and oxidised (GSSG) glutathione by upregulating gamma-glutamylcysteine ligase (γ-GCL) involved in GSH biosynthesis and inhibiting the activities of redox enzymes responsible for GSH utilization and recycling, such as glutathione-S-transferase (GST) and glutathione reductase (GR). Molecular docking analysis suggests putative interactions of DISPOL with GST and GR which could account for its inhibitory effect on these enzymes. Further, DISPOL induced reductive environment preceded G1 arrest and apoptosis as evidenced by decreased expression of cell cycle genes (Cyclin D1 and Cyclin E1) and elevation of p21 and apoptotic markers (cleaved caspase 3 and cleaved PARP). The combinatorial experiments involving DISPOL and redox modulatory agents such as N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) indeed confirmed the role of reductive stress in DISPOL-induced cell death. Finally, Lipinski's rule suggests attributes of drug likeness in DISPOL. Taken together, DISPOL exhibits a novel mechanism of reductive stress-mediated cell death in A549 cells that warrants future exploration as anticancer agent.

Novel molecular insights into the anti-oxidative stress response and structure-function of a salt-inducible cyanobacterial Mn-catalase.

KatB, a salt-inducible Mn-catalase, protects the cyanobacterium Anabaena from salinity/oxidative stress. In this report, we provide distinctive insights into the biological-biochemical function of KatB at the molecular level. Anabaena overexpressing the wild-type KatB protein (KatBWT) detoxified H2 O2 efficiently, showing reduced burden of reactive oxygen species compared with the strain overproducing KatBF2V (wherein F-2 is replaced by V). Correspondingly, the KatBWT protein also displayed several folds more activity than KatBF2V. Interestingly, the KatB variants with large hydrophobic amino acids (F/W/Y) were more compact, showed enhanced activity, and were resistant to thermal/chemical denaturation than variants with smaller residues (G/A/V) at the second position. X-ray crystallography-based analysis showed that F-2 was required for appropriate interactions between two subunits. These contacts provided stability to the hexamer, making it more compact. F-2, through its interaction with F-66 and W-43, formed the proper hydrophobic pocket that held the active site together. Consequently, only residues that supported activity (i.e., F/Y/W) were selected at the second position in Mn-catalases during evolution. This study (a) demonstrates that modification of nonactive site residues can alter the response of catalases to environmental stress and (b) has expanded the scope of amino acids that can be targeted for rational protein engineering in plants.

Phosphorylation of Deinococcus radiodurans RecA Regulates Its Activity and May Contribute to Radioresistance.

Deinococcus radiodurans has a remarkable capacity to survive exposure to extreme levels of radiation that cause hundreds of DNA double strand breaks (DSBs). DSB repair in this bacterium depends on its recombinase A protein (DrRecA). DrRecA plays a pivotal role in both extended synthesis-dependent strand annealing and slow crossover events of DSB repair during the organism's recovery from DNA damage. The mechanisms that control DrRecA activity during the D. radiodurans response to γ radiation exposure are unknown. Here, we show that DrRecA undergoes phosphorylation at Tyr-77 and Thr-318 by a DNA damage-responsive serine threonine/tyrosine protein kinase (RqkA). Phosphorylation modifies the activity of DrRecA in several ways, including increasing its affinity for dsDNA and its preference for dATP over ATP. Strand exchange reactions catalyzed by phosphorylated versus unphosphorylated DrRecA also differ. In silico analysis of DrRecA structure support the idea that phosphorylation can modulate crucial functions of this protein. Collectively, our findings suggest that phosphorylation of DrRecA enables the recombinase to selectively use abundant dsDNA substrate present during post-irradiation recovery for efficient DSB repair, thereby promoting the extraordinary radioresistance of D. radiodurans.

A Salt-Inducible Mn-Catalase (KatB) Protects Cyanobacterium from Oxidative Stress.

Catalases, enzymes that detoxify H2O2, are widely distributed in all phyla, including cyanobacteria. Unlike the heme-containing catalases, the physiological roles of Mn-catalases remain inadequately characterized. In the cyanobacterium Anabaena, pretreatment of cells with NaCl resulted in unusually enhanced tolerance to oxidative stress. On exposure to H2O2, the NaCl-treated Anabaena showed reduced formation of reactive oxygen species, peroxides, and oxidized proteins than the control cells (i.e. not treated with NaCl) exposed to H2O2. This protective effect correlated well with the substantial increase in production of KatB, a Mn-catalase. Addition of NaCl did not safeguard the katB mutant from H2O2, suggesting that KatB was indeed responsible for detoxifying the externally added H2O2. Moreover, Anabaena deficient in KatB was susceptible to oxidative effects of salinity stress. The katB gene was strongly induced in response to osmotic stress or desiccation. Promoter-gfp analysis showed katB to be expressed only in the vegetative cells but not in heterocysts. Biochemically, KatB was an efficient, robust catalase that remained active in the presence of high concentrations of NaCl. Our findings unravel the role of Mn-catalase in acclimatization to salt/oxidative stress and demonstrate that the oxidative stress resistance of an organism can be enhanced by a simple compound such as NaCl.

Unveiling potential inhibitors targeting the nucleocapsid protein of SARS-CoV-2: Structural insights into their binding sites.

The Nucleocapsid (N) protein of SARS-CoV-2 plays a crucial role in viral replication and pathogenesis, making it an attractive target for developing antiviral therapeutics. In this study, we used differential scanning fluorimetry to establish a high-throughput screening method for identifying high-affinity ligands of N-terminal domain of the N protein (N-NTD). We screened an FDA-approved drug library of 1813 compounds and identified 102 compounds interacting with N-NTD. The screened compounds were further investigated for their ability to inhibit the nucleic-acid binding activity of the N protein using electrophoretic mobility-shift assays. We have identified three inhibitors, Ceftazidime, Sennoside A, and Tannic acid, that disrupt the N protein's interaction with RNA probe. Ceftazidime and Sennoside A exhibited nano-molar range binding affinities with N protein, determined through surface plasmon resonance. The binding sites of Ceftazidime and Sennoside A were investigated using [1H, 15N]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. Ceftazidime and Sennoside A bind to the putative RNA binding site of the N protein, thus providing insights into the inhibitory mechanism of these compounds. These findings will contribute to the development of novel antiviral agents targeting the N protein of SARS-CoV-2.

Purification, crystallization and preliminary crystallographic analysis of KatB, a manganese catalase from Anabaena PCC 7120.

Catalases are enzymes that play an important role in the detoxification of hydrogen peroxide (H2O2) in aerobic organisms. Among catalases, haem-containing catalases are ubiquitously distributed and their enzymatic mechanism is very well understood. On the other hand, manganese catalases that contain a bimanganese core in the active site have been less well characterized and their mode of action is not fully understood. The genome of Anabaena PCC 7120 does not show the presence of a haem catalase-like gene; instead, two ORFs encoding manganese catalases (Mn-catalases) are present. Here, the crystallization and preliminary X-ray crystallographic analysis of KatB, one of the two Mn-catalases from Anabaena, are reported. KatB was crystallized using the hanging-drop vapour-diffusion method with PEG 400 as a precipitant and calcium acetate as an additive. Diffraction data were collected in-house on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 2.2 Šresolution at 100 K. The tetragonal crystal belonged to space group P4(1)2(1)2 (or enantiomer), with unit-cell parameters a = b = 101.87, c = 138.86 Å. Preliminary X-ray diffraction analysis using the Matthews coefficient and self-rotation function suggests the presence of a trimer in the asymmetric unit.

Molecular basis for reduced cleavage activity and drug resistance in D30N HIV-1 protease.

Nelfinavir is one of the FDA-approved HIV-1 protease inhibitors and a part of highly active anti-retroviral therapy (HAART) for the treatment of HIV-AIDS. Nelfinavir was the first HIV-1 protease inhibitor to be approved as a paediatric formulation. The application of HAART had resulted in significant improvement in the lives of AIDS patients. However, the emergence of drug resistance in HIV-1 protease has limited the use of many of these drugs including nelfinavir. A unique mutation observed frequently in patients treated with nelfinavir is D30N as it is selected exclusively by nelfinavir. The D30N mutation imparts very high resistance to nelfinavir but unlike other primary mutations does not give cross-resistance to the majority of other drugs. D30N mutation also significantly reduces cleavage activity of HIV-1 protease and affects viral fitness. Here, we have determined crystal structures of D30N HIV-1 protease in unliganded form and in complex with nelfinavir. These structures provide the rationale for reduced cleavage activity and the molecular basis of drug resistance induced by D30N mutation. The loss of coulombic interaction part of a crucial hydrogen bond between the drug and the protease is likely to play a major role in reduced affinity and resistance towards nelfinavir. The decreased catalytic activity of D30N HIV-1 protease due to altered interaction with the substrates and reduced stability of folding core may be the reason for the reduced replicative capacity of the virus harboring mutant HIV-1 protease.Communicated by Ramaswamy H. Sarma.

Unique functional insights into the antioxidant response of the cyanobacterial Mn-catalase (KatB).

KatB, a hexameric Mn-catalase, plays a vital role in overcoming oxidative and salinity stress in the ecologically important, N2-fixing cyanobacterium, Anabaena. The 5 N-terminal residues of KatB, which show a high degree of conservation in cyanobacteria, form an antiparallel ß-strand at the subunit interface of the KatB hexamer. In this study, the contribution of these N-terminal non-active site residues, towards the maintenance of the structure, biochemical properties, and redox balance was evaluated. Each N-terminal amino acid residue from the 2nd to the 7th position of KatB was individually mutated to Ala (to express KatBF2A/KatBF3A/KatBH4A/KatBK5E/KatBK6A/KatBE7A) or this entire 6 amino acid stretch was deleted (to yield KatBTrunc). All the above-mentioned KatB variants, along with the wild-type KatB protein (KatBWT), were overproduced in E. coli and purified. In comparison to KatBWT, the KatBF2A/KatBH4A/KatBTrunc proteins were less compact, more prone to chemical/thermal denaturation, and were unexpectedly inactive. KatBF3A/KatBK5E/KatBK6A showed biophysical/biochemical properties that were in between that of KatBWT and KatBF2A/KatBH4A/KatBTrunc. Surprisingly, KatBE7A was more thermostable with higher activity than KatBWT. On exposure to H2O2, E. coli expressing KatBWT/KatBE7A showed considerably reduced formation of ROS and increased survival than the other KatB variants. Utilizing the KatB structure, the molecular basis responsible for the altered stability/activity of the KatB mutants was delineated. This study demonstrates the physiological importance of the N-terminal ß-strand of Mn-catalases in combating H2O2 stress and shows that the non-active site residues can be used for rational protein engineering to develop Mn-catalases with improved characteristics.

WD40 domain of RqkA regulates its kinase activity and role in extraordinary radioresistance of D. radiodurans.

RqkA, a DNA damage responsive serine/threonine kinase, is characterized for its role in DNA repair and cell division in D. radiodurans. It has a unique combination of a kinase domain at N-terminus and a WD40 type domain at C-terminus joined through a linker. WD40 domain is comprised of eight ß-propeller repeats held together via 'tryptophan-docking motifs' and forming a typical 'velcro' closure structure. RqkA mutants lacking the WD40 region (hereafter referred to as WD mutant) could not complement RqkA loss in γ radiation resistance in D. radiodurans and lacked γ radiation-mediated activation of kinase activity in vivo. WD mutants failed to phosphorylate its cognate substrate (e.g. DrRecA) in surrogate E. coli cells. Unlike wild-type enzyme, the kinase activity of its WD40 mutants was not stimulated by pyrroloquinoline quinine (PQQ) indicating the role of the WD motifs in PQQ interaction and stimulation of its kinase activity. Together, results highlighted the importance of the WD40 domain in the regulation of RqkA kinase signaling functions in vivo, and thus, the role of WD40 domain in the regulation of any STPK is first time demonstrated in bacteria.Communicated by Ramaswamy H. Sarma.

Structural insights into SARS-CoV-2 proteins.

The unprecedented scale of the ongoing COVID-19 pandemic has catalyzed an intense effort of the global scientific community to unravel different aspects of the disease in a short time. One of the crucial aspects of these developments is the determination of more than three hundred experimental structures of SARS-CoV-2 proteins in the last few months. These include structures of viral non-structural, structural, and accessory proteins and their complexes determined by either X-ray diffraction or cryo-electron microscopy. These structures elucidate the intricate working of different components of the viral machinery at the atomic level during different steps of the viral life cycle, including attachment to the host cell, viral genome replication and transcription, and genome packaging and assembly of the virion. Some of these proteins are also potential targets for drug development against the disease. In this review, we discuss important structural features of different SARS-CoV-2 proteins with their function, and their potential as a target for therapeutic interventions.

X-ray snapshot of HIV-1 protease in action: observation of tetrahedral intermediate and short ionic hydrogen bond SIHB with catalytic aspartate.

Structural snapshots of each step in the catalytic cycle would help development of inhibitors of human immunodeficiency virus type 1 protease (HIV-1 PR) as effective drugs against HIV/AIDS. We report here one snapshot obtained by determining the structure of enzyme-substrate complex under conditions where the catalytic activity of the enzyme is greatly reduced. The 1.76 A crystal structure shows the oligopeptide substrate, AETFYVDGAA, converted in situ into a gem-diol tetrahedral intermediate (TI). The gem-diol intermediate is neutral and one of the hydroxyl oxygens forms a very short hydrogen bond (2.2 A) with the anionic aspartate of the catalytic dyad, which is monoprotonated. Further, there is no hydrogen atom on the outer oxygen of the neutral aspartate. These two observations provide direct evidence that, in the reaction mechanism, hydrogen bonding between catalytic aspartate and scissile carbonyl oxygen facilitates water attack on the scissile carbon atom. Comparison with the structural snapshot of the biproduct complex involving the same substrate reveals the reorganization of the hydrogen bonds at the catalytic center as the enzymatic reaction progresses toward completion. Accumulation of TI in the crystals provides direct evidence that collapse of TI is the rate-limiting step of hydrolysis.

Insights into the mechanism of drug resistance: X-ray structure analysis of G48V/C95F tethered HIV-1 protease dimer/saquinavir complex.

The mutation G48V in HIV-1 protease is a major resistance mutation against the drug saquinavir. Recently, G48V mutation is found to co-exist with the mutation C95F in AIDS patients treated with saquinavir. We report here the three-dimensional crystal structure of G48V/C95F tethered HIV-1 protease/saquinavir complex. The structure indicates following as the possible causes of drug resistance: (1) loss of direct van der Waals interactions between saquinavir and enzyme residues PHE-53 and PRO-1081, (2) loss of water-mediated hydrogen bonds between the carbonyl oxygen atoms in saquinavir and amide nitrogen atoms of flap residues 50 and 1050, (3) changes in inter-monomer interactions, which could affect the energetics of domain movements associated with inhibitor-binding, and (4) significant reduction in the stability of the mutant dimer. The present structure also provides a rationale for the clinical observation that the resistance mutations C95F/G48V/V82A occur as a cluster in AIDS patients.

Gazing into the remarkable world of non-heme catalases through the window of the cyanobacterial Mn-catalase 'KatB'.

Catalases, enzymes that decompose H2O2, are broadly categorized as heme catalases or non-heme catalases. The non-heme catalases are also known as Mn-catalases as they have Mn atoms in their active sites. However, unlike the well characterized heme-catalases, the study of Mn-catalases has gained importance only in the last few years. The filamentous, heterocystous, N2-fixing cyanobacterium Anabaena PCC 7120, shows the presence of two Mn-catalases, KatA and KatB, but lacks heme catalases. Of the two Mn-catalases, KatB, which is induced by salt/desiccation, plays a major role in overcoming salinity/oxidative stress. In this mini review, we have summarized the recent advances made in the field of Mn-catalases, particularly KatB, and have interpreted these results in the larger context of stress physiology. These aspects bring to the fore the distinctive biochemical/structural properties of Mn-catalases and furthermore highlight the in vivo importance of these enzymes in adapting to oxidative stresses.

Pharmacological characterization of a structurally new class of antibacterial compound, triphenyl-phosphonium conjugated diarylheptanoid: Antibacterial activity and molecular mechanism.

Many pathogenic species of bacteria are showing increasing drug resistance against clinically used antibiotics. Molecules structurally distant from known antibiotics and possessing membrane targeting bactericidal activities are more likely to display activity against drug-resistant pathogens. Mitocurcumin (MitoC) is one of such compounds, synthesized by triphenyl-phosphonium conjugation with curcumin, and has been shown recently from our laboratory to have broad-spectrum bactericidal activity (Kumari et al. 2019 Free Radic. Biol. Med. 143, 140-145). Here, we further demonstrate the antibacterial properties of MitoC against resistant strains and also its mechanism of action. It displays efficient bactericidal activity against multidrug-resistant Staphylococcus aureus and Streptococcus pneumoniae (MIC values in the 1.5-12.5 µM range), and coagulase-negative Staphylococci do not show resistance development against MitoC. Liposome based studies and MIC values against TolC deletion mutant (Δ tolC; outer membrane protein) of E. coli suggest extensive membrane damage to be the primary mechanism of bactericidal activity. MitoC did not exhibit toxicity in BALB/c mice with an oral administration of 250 mg/kg body weight and was found to be totally safe without any significant effect on haematological, biochemical parameters and inflammatory responses. Its rapid bactericidal action as assessed by in vitro time-kill assay against B. subtilis, compared to ciprofloxacin, and long half-life in rodent serum, suggest that MitoC could be an excellent lead-molecule against drug-resistant pathogens. The highlights of the study are that mitocurcumin belongs to a structurally new class of bactericidal compounds. It displays activity against MDR strains of pathogenic bacteria and challenging MRSA. Liposome-based studies confirm the membrane damaging property of the molecule. Mitocurcumin does not show resistance development even after 27 bacterial generations.

X-ray structure of HIV-1 protease in situ product complex.

HIV-1 protease is an effective target for design of different types of drugs against AIDS. HIV-1 protease is also one of the few enzymes that can cleave substrates containing both proline and nonproline residues at the cleavage site. We report here the first structure of HIV-1 protease complexed with the product peptides SQNY and PIV derived by in situ cleavage of the oligopeptide substrate SQNYPIV, within the crystals. In the structure, refined against 2.0-A resolution synchrotron data, a carboxyl oxygen of SQNY is hydrogen-bonded with the N-terminal nitrogen atom of PIV. At the same time, this proline nitrogen atom does not form any hydrogen bond with catalytic aspartates. These two observations suggest that the protonation of scissile nitrogen, during peptide bond cleavage, is by a gem-hydroxyl of the tetrahedral intermediate rather than by a catalytic aspartic acid.

Resistance mechanism revealed by crystal structures of unliganded nelfinavir-resistant HIV-1 protease non-active site mutants N88D and N88S.

Nelfinavir is an inhibitor of HIV-1 protease, and is used for treatment of patients suffering from HIV/AIDS. However, treatment results in drug resistant mutations in HIV-1 protease. N88D and N88S are two such mutations which occur in the non-active site region of the enzyme. We have determined crystal structures of unliganded N88D and N88S mutants of HIV-1 protease to resolution of 1.65A and 1.8A, respectively. These structures refined against synchrotron data lead to R-factors of 0.1859 and 0.1780, respectively. While structural effects of N88D are very subtle, the mutation N88S has caused a significant conformational change in D30, an active site residue crucial for substrate and inhibitor binding.

Crystallization and preliminary X-ray crystallographic analysis of PhoK, an extracellular alkaline phosphatase from Sphingomonas sp. BSAR-1.

Alkaline phosphatases (APs) are widely distributed from microbes to humans and are involved in several important biological processes such as phosphate nutrition, signal transduction and pathogenesis. Alkaline phosphatases are also useful in various industrial applications and in recombinant DNA technology. A new AP enzyme from Sphingomonas sp. strain BSAR-1, termed PhoK, has been shown to be useful in uranium bioprecipitation. PhoK was expressed, purified and crystallized. The crystals belonged to space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 87.37, c = 168.16 A, and contained one enzyme molecule in the asymmetric unit. Native diffraction data have been collected to 1.95 A resolution at the ESRF.