Brevicillin, a novel lanthipeptide from the genus Brevibacillus with antimicrobial, antifungal, and antiviral activity.

AIM: This study was aimed to determine antimicrobial and antiviral activity of a novel lanthipeptide from a Brevibacillus sp. for disinfectant application. METHODS AND RESULTS: The antimicrobial peptide (AMP) was produced by a bacterial strain AF8 identified as a member of the genus Brevibacillus representing a novel species. Whole genome sequence analysis using BAGEL identified a putative complete biosynthetic gene cluster involved in lanthipeptide synthesis. The deduced amino acid sequence of lanthipeptide named as brevicillin, showed >30% similarity with epidermin. Mass determined by MALDI-MS and Q-TOF suggested posttranslational modifications like dehydration of all Ser and Thr amino acids to yield Dha and Dhb, respectively. Amino acid composition determined upon acid hydrolysis is in agreement with core peptide sequence deduced from the putative biosynthetic gene bvrAF8. Biochemical evidence along with stability features ascertained posttranslational modifications during formation of the core peptide. The peptide showed strong activity with 99% killing of pathogens at 12 µg ml-1 within 1 minute. Interestingly, it also showed potent anti-SARS-CoV-2 activity by inhibiting ∼99% virus growth at 10 µg ml-1 in cell culture-based assay. Brevicillin did not show dermal allergic reactions in BALB/c mice. CONCLUSION: This study provides detailed description of a novel lanthipeptide and demonstrates its effective antibacterial, antifungal and anti-SARS-CoV-2 activity.

Molecular docking and dynamic simulation approach to decipher steroidal sapogenins (genus Trillium) derived agonists for glucocorticoid receptor.

Steroidal sapogenins (SS) are structural analogues of steroidal drugs, which are frequently used for the treatment of several diseases including reproductive, malignancies, neurological, and inflammation-related diseases. The glucocorticoid receptor (GR) is a nuclear receptor that regulates development, metabolism, and inflammation, in response to steroidal ligands. Therefore, GR is considered as a potential therapeutic target for steroidal agents to the treatment of inflammation-related diseases. We hypothesized that SS may act as an agonist for GR due to structural similarity with corticosteroids. In this study, we carried out in silico screening of various SS from the genus Trillium to check their potential as an agonist for GR. Our data suggest that out of 42 SS, only 7 molecules have interacted with GR. However, molecular mechanics with generalized Born and surface area (MM-GBSA) analysis revealed that only two SS (SS 38 and SS 39) molecules bind favorably to GR. Among these, SS 38 (docking score: -9.722 Kcal/mol and MM-GBSA ΔGbind: -50.192 Kcal/mol) and SS 39 (docking score: -11.20 Kcal/mol and MM-GBSA ΔGbind: -58.937 Kcal/mol) have best docking and MM-GBSA scores. Molecular dynamics (MD) simulation studies of SS 38, SS 39, and dexamethasone-GR complex revealed that both SS shows hydrogen bonding and hydrophobic interaction with GR over the 120 ns simulation with mild fluctuations. The current study suggests that SS 38 and SS 39 may be further explored as a potential agonist to treat several disease conditions mediated by GR.

Identification of small molecules targeting homoserine acetyl transferase from Mycobacterium tuberculosis and Staphylococcus aureus.

There is an urgent need to validate new drug targets and identify small molecules that possess activity against both drug-resistant and drug-sensitive bacteria. The enzymes belonging to amino acid biosynthesis have been shown to be essential for growth in vitro, in vivo and have not been exploited much for the development of anti-tubercular agents. Here, we have identified small molecule inhibitors targeting homoserine acetyl transferase (HSAT, MetX, Rv3341) from M. tuberculosis. MetX catalyses the first committed step in L-methionine and S-adenosyl methionine biosynthesis resulting in the formation of O-acetyl-homoserine. Using CRISPRi approach, we demonstrate that conditional repression of metX resulted in inhibition of M. tuberculosis growth in vitro. We have determined steady state kinetic parameters for the acetylation of L-homoserine by Rv3341. We show that the recombinant enzyme followed Michaelis-Menten kinetics and utilizes both acetyl-CoA and propionyl-CoA as acyl-donors. High-throughput screening of a 2443 compound library resulted in identification of small molecule inhibitors against MetX enzyme from M. tuberculosis. The identified lead compounds inhibited Rv3341 enzymatic activity in a dose dependent manner and were also active against HSAT homolog from S. aureus. Molecular docking of the identified primary hits predicted residues that are essential for their binding in HSAT homologs from M. tuberculosis and S. aureus. ThermoFluor assay demonstrated direct binding of the identified primary hits with HSAT proteins. Few of the identified small molecules were able to inhibit growth of M. tuberculosis and S. aureus in liquid cultures. Taken together, our findings validated HSAT as an attractive target for development of new broad-spectrum anti-bacterial agents that should be effective against drug-resistant bacteria.

Potential Inhibitors of SARS-CoV-2 Main Protease (Mpro) Identified from the Library of FDA-Approved Drugs Using Molecular Docking Studies.

The Corona Virus Infectious Disease-2019 (COVID-19) outbreak originated at Wuhan, China, in December 2019. It has already spread rapidly and caused more than 6.5 million deaths worldwide. Its causal agent is a beta-coronavirus named SARS-CoV-2. Many efforts have already been made to develop new vaccines and drugs against these viruses, but over time, it has changed its molecular nature and evolved into more lethal variants, such as Delta and Omicron. These will lead us to target its more-conserved proteins. The sequences' BLAST and crystal structure of the main protease Mpro suggest a high sequence and structural conservation. Mpro is responsible for the proteolytic maturation of the polyprotein essential for the viral replication and transcription, which makes it an important drug target. Discovery of new drug molecules may take years before getting to the clinics. So, considering urgency, we performed molecular docking studies using FDA-approved drugs to identify molecules that could potentially bind to the substrate-binding site and inhibit SARS-CoV-2's main protease (Mpro). We used the Glide module in the Schrödinger software suite to perform molecular docking studies, followed by MM-GBSA-based energy calculations to score the hit molecules. Molecular docking and manual analysis suggest that several drugs may bind and potentially inhibit Mpro. We also performed molecular simulations studies for selected compounds to evaluate protein-drug interactions. Considering bioavailability, lesser toxicity, and route of administration, some of the top-ranked drugs, including lumefantrine (antimalarial), dipyridamole (coronary vasodilator), dihydroergotamine (used for treating migraine), hexoprenaline (anti asthmatic), riboflavin (vitamin B2), and pantethine (vitamin B5) may be taken forward for further in vitro and in vivo experiments to investigate their therapeutic potential.

Structural insights into the mechanism of Type IVa pilus extension and retraction ATPase motors.

Type IVa pili are bacterial appendages involved in diverse physiological processes, including electron transfer in Geobacter sulfurreducens. ATP hydrolysis coupled with conformational changes powers the extension (PilB) and retraction (PilT) motors in the pilus machinery. We report the unliganded crystal structures of the core ATPase domain of PilB and PilT-4 from G. sulfurreducens at 3.1 and 2.6 Å resolution, respectively. PilB structure revealed three distinct conformations, that is, open, closed, and open' which were previously proposed to be mediated by ATP/ADP binding. PilT-4 subunits, on the other hand, were observed in the closed state conformation. We further report that both PilB and PilT-4 hexamers have two high-affinity ATP-binding sites. Comparative structural analysis and solution data presented here supports the "symmetric rotary model" for these ATPase motors. Our data further suggest that pores of these motors rotate either clockwise or counterclockwise to facilitate assembly or disassembly of right-handed or left-handed pilus. DATABASE: Structural data are available in the RCSB PDB database under the PDB ID 5ZFQ (PilT-4), 5ZFR (PilB).

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food.

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages.

Dual Role of a Biosynthetic Enzyme, CysK, in Contact Dependent Growth Inhibition in Bacteria.

Contact dependent growth inhibition (CDI) is the phenomenon where CDI+ bacterial strain (inhibitor) inhibits the growth of CDI-strain (target) by direct cell to cell contact. CDI is mediated by cdiBAI gene cluster where CdiB facilitates the export of CdiA, an exotoxin, on the cell surface and CdiI acts as an immunity protein to protect CDI+ cells from autoinhibition. CdiA-CT, the C-terminal region of the toxin CdiA, from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires binding of a biosynthetic enzyme CysK (O-acetylserine sulfyhydrylase) for activation in the target cells. CdiA-CT can also interact simultaneously with CysK and immunity protein, CdiI, to form a ternary complex in UPEC536. But the role of CysK in the ternary complex is not clear. We studied the hydrodynamic, thermodynamic and kinetic parameters of binary and ternary complexes using AUC, ITC and SPR respectively, to investigate the role of CysK in UPEC536. We report that CdiA-CT binds CdiI and CysK with nanomolar range affinity. We further report that binding of CysK to CdiA-CT improves its affinity towards CdiI by ~40 fold resulting in the formation of a more stable complex with over ~130 fold decrease in dissociation rate. Thermal melting experiments also suggest the role of CysK in stabilizing CdiA-CT/CdiI complex as Tm of the binary complex shifts ~10°C upon binding CysK. Hence, CysK acts a modulator of CdiA-CT/CdiI interactions by stabilizing CdiA-CT/CdiI complex and may play a crucial role in preventing autoinhibition in UPEC536. This study reports a new moonlighting function of a biosynthetic enzyme, CysK, as a modulator of toxin/immunity interactions in UPEC536 inhibitor cells.

The intramolecular disulfide-stapled structure of laterosporulin, a class IId bacteriocin, conceals a human defensin-like structural module.

The growing emergence of antibiotic-resistant bacteria has led to the exploration of naturally occurring defense peptides as antimicrobials. In this study, we found that laterosporulin (LS), a class IId bacteriocin, effectively kills active and nonmultiplying cells of both Gram-positive and Gram-negative bacteria. Fluorescence and electron microscopy suggest that growth inhibition occurs because of increased membrane permeability. The crystal structure of LS at 2.0 Å resolution reveals an all-ß conformation of this peptide, with four ß-strands forming a twisted ß-sheet. All six intrinsic cysteines are intramolecularly disulfide-bonded, with two disulfides constraining the N terminus of the peptide and the third disulfide crosslinking the extreme C terminus, resulting in the formation of a closed structure. The significance of disulfides in maintaining the in-solution peptide structure was confirmed by CD and fluorescence analyses. Despite a low overall sequence similarity, LS has disulfide connectivity [C(I)-C(V), C(II)-C(IV), and C(III)-C(VI)] like that of ß-defensins and a striking architectural similarity with α-defensins. Therefore LS presents a missing link between bacteriocins and mammalian defensins, and is also a potential antimicrobial lead, in particular against nonmultiplying bacteria.

Mycobacterium tuberculosis keto-mycolic acid and macrophage nuclear receptor TR4 modulate foamy biogenesis in granulomas: a case of a heterologous and noncanonical ligand-receptor pair.

The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA-TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.

Structural basis for the redox sensitivity of the Mycobacterium tuberculosis SigK-RskA σ-anti-σ complex.

The host-pathogen interactions in Mycobacterium tuberculosis infection are significantly influenced by redox stimuli and alterations in the levels of secreted antigens. The extracytoplasmic function (ECF) σ factor σ(K) governs the transcription of the serodominant antigens MPT70 and MPT83. The cellular levels of σ(K) are regulated by the membrane-associated anti-σ(K) (RskA) that localizes σ(K) in an inactive complex. The crystal structure of M. tuberculosis σ(K) in complex with the cytosolic domain of RskA (RskAcyto) revealed a disulfide bridge in the -35 promoter-interaction region of σ(K). Biochemical experiments reveal that the redox potential of the disulfide-forming cysteines in σ(K) is consistent with its role as a sensor. The disulfide bond in σ(K) influences the stability of the σ(K)-RskAcyto complex but does not interfere with σ(K)-promoter DNA interactions. It is noted that these disulfide-forming cysteines are conserved across homologues, suggesting that this could be a general mechanism for redox-sensitive transcription regulation.

Structural and biochemical bases for the redox sensitivity of Mycobacterium tuberculosis RslA.

An effective transcriptional response to redox stimuli is of particular importance for Mycobacterium tuberculosis, as it adapts to the environment of host alveoli and macrophages. The M. tuberculosis sigma factor sigma(L) regulates the expression of genes involved in cell-wall and polyketide syntheses. sigma(L) interacts with the cytosolic anti-sigma domain of a membrane-associated protein, RslA. Here we demonstrate that RslA binds Zn(2+) and can sequester sigma(L) in a reducing environment. In response to an oxidative stimulus, proximal cysteines in the CXXC motif of RslA form a disulfide bond, releasing bound Zn(2+). This results in a substantial rearrangement of the sigma(L)/RslA complex, leading to an 8-fold decrease in the affinity of RslA for sigma(L). The crystal structure of the -35-element recognition domain of sigma(L), sigma(4)(L), bound to RslA reveals that RslA inactivates sigma(L) by sterically occluding promoter DNA and RNA polymerase binding sites. The crystal structure further reveals that the cysteine residues that coordinate Zn(2+) in RslA are solvent exposed in the complex, thus providing a structural basis for the redox sensitivity of RslA. The biophysical parameters of sigma(L)/RslA interactions provide a template for understanding how variations in the rate of Zn(2+) release and associated conformational changes could regulate the activity of a Zn(2+)-associated anti-sigma factor.

X-ray crystallographic and NMR studies of pantothenate synthetase provide insights into the mechanism of homotropic inhibition by pantoate.

The structural basis for the homotropic inhibition of pantothenate synthetase by the substrate pantoate was investigated by X-ray crystallography and high-resolution NMR spectroscopic methods. The tertiary structure of the dimeric N-terminal domain of Escherichia coli pantothenate synthetase, determined by X-ray crystallography to a resolution of 1.7 A, showed a second molecule of pantoate bound in the ATP-binding pocket. Pantoate binding to the ATP-binding site induced large changes in structure, mainly for backbone and side chain atoms of residues in the ATP binding HXGH(34-37) motif. Sequence-specific NMR resonance assignments and solution secondary structure of the dimeric N-terminal domain, obtained using samples enriched in (2)H, (13)C, and (15)N, indicated that the secondary structural elements were conserved in solution. Nitrogen-15 edited two-dimensional solution NMR chemical shift mapping experiments revealed that pantoate, at 10 mm, bound at these two independent sites. The solution NMR studies unambiguously demonstrated that ATP stoichiometrically displaced pantoate from the ATP-binding site. All NMR and X-ray studies were conducted at substrate concentrations used for enzymatic characterization of pantothenate synthetase from different sources [Jonczyk R & Genschel U (2006) J Biol Chem 281, 37435-37446]. As pantoate binding to its canonical site is structurally conserved, these results demonstrate that the observed homotropic effects of pantoate on pantothenate biosynthesis are caused by competitive binding of this substrate to the ATP-binding site. The results presented here have implications for the design and development of potential antibacterial and herbicidal agents.