Enzymatic reconstitution of salicylate formation in promysalin biosynthesis.

Promysalin is an amphipathic antibiotic isolated from Pseudomonas promysalinigenes (previously Pseudomonas putida RW10S1) which shows potent antibacterial activities against Gram-negative pathogens by inactivating succinate dehydrogenase. Based on the in-vivo studies, promysalin is hypothesized to be assembled from three building blocks: salicylic acid, proline, and myristic acid via a proposed but uncharacterized hybrid NRPS-PKS biosynthetic pathway. So far, no in-vitro biosynthetic studies have been reported for this promising antibiotic. Here, we report the first in-vitro reconstitution and biochemical characterization of two early enzymes on the pathway: PpgH, an isochorismate synthase (IS), and PpgG, an isochorismate pyruvate lyase (IPL) which are involved in the biosynthesis of salicylic acid, the polar fragment of promysalin. We also report a secondary chorismate mutase (CM) activity for PpgG. Based on our biochemical experiments, preliminary mechanistic proposals have been postulated for PpgH and PpgG. We believe this study will lay a strong foundation for elucidating the functions and mechanisms of other intriguing enzymes of the promysalin biosynthesis pathway, which may potentially unravel interesting enzyme chemistries and promote pathway engineering in the future.

Gene expression and molecular characterization of recombinant subtilisin from Bacillus subtilis with antibacterial, antioxidant and anticancer properties.

This study investigated the multifunctional attributes such as, antibacterial, antioxidant and anticancer potential of recombinant subtilisin. A codon-optimized subtilisin gene was synthesized from Bacillus subtilis and was successfully transformed into E. coli DH5α cells which was further induced for high level expression in E. coli BL21 (DE3). An affinity purified ~40 kDa recombinant subtilisin was obtained that revealed to be highly alkali-thermostable based on the thermodynamic parameters. The kinetic parameters were deduced that indicated higher affinity of N-Suc-F-A-A-F-pNA substrate towards subtilisin. Recombinant subtilisin demonstrated strong antibacterial activity against several pathogens and showed minimum inhibitory concentration of 0.06 µg/mL against B. licheniformis and also revealed high stability under the influence of several biochemical factors. It also displayed antioxidant potential in a dose dependent manner and exhibited cell cytotoxicity against A549 and MCF-7 cancerous cell lines with IC50 of 5 µM and 12 µM respectively. The identity of recombinant subtilisin was established by MALDI-TOF mass spectrum depicting desired mass peaks and N-terminal sequence as MRSK by MALDI-TOF-MS. The deduced N- terminal amino acid sequence by Edman degradation revealed high sequence similarity with subtilisins from Bacillus strains. The structural and functional analysis of recombinant antibacterial subtilisin was elucidated by Raman, circular dichroism and nuclear magnetic resonance spectroscopy and thermogravimetric analysis. The results contribute to the development of highly efficient subtilisin with enhanced catalytic properties making it a promising candidate for therapeutic applications in healthcare industries.

Unfolding of the SARS-CoV-2 spike protein through infrared and ultraviolet-C radiation based disinfection.

The spreading of coronavirus from contacting surfaces and aerosols created a pandemic around the world. To prevent the transmission of SARS-CoV-2 virus and other contagious microbes, disinfection of contacting surfaces is necessary. In this study, a disinfection box equipped with infrared (IR) radiation heating and ultraviolet-C (UV-C) radiation is designed and tested for its disinfection ability against pathogenic bacteria and SARS-CoV-2 spike protein. The killing of a Gram-positive, namely, S. aureus and a Gram-negative namely, S. typhi bacteria was studied followed by the inactivation of the spike protein. The experimental parameters were optimized using a statistical tool. For the broad-spectrum antibacterial activity, the optimum condition was holding at 65.61 °C for 13.54 min. The killing of the bacterial pathogen occurred via rupturing the cell walls as depicted by electron microscopy. Further, the unfolding of SARS-CoV-2 spike protein and RNase A was studied under IR and UV-C irradiations at the aforesaid optimized condition. The unfolding of both the proteins was confirmed by changes in the secondary structure, particularly an increase in ß-sheets and a decrease in α-helixes. Remarkably, the higher penetration depth of IR waves up to subcutaneous tissue resulted in lower optimum disinfection temperature, <70 °C in vogue. Thus, the combined UV-C and IR radiation is effective in killing the pathogenic bacteria and denaturing the glycoproteins.

Gene Expression and Characterization of Iturin A Lipopeptide Biosurfactant from Bacillus aryabhattai for Enhanced Oil Recovery.

Biosurfactants are eco-friendly surface-active molecules recommended for enhanced oil recovery techniques. In the present study, a potential lipopeptide (biosurfactant) encoding the iturin A gene was synthesized from Bacillus aryabhattai. To improvise the yield of the lipopeptide for specific applications, current research tends toward engineering and expressing recombinant peptides. An iturin A gene sequence was codon-optimized, amplified with gene-specific primers, and ligated into the pET-32A expression vector to achieve high-level protein expression. The plasmid construct was transformed into an E. coli BL21 DE3 host to evaluate the expression. The highly expressed recombinant iturin A lipopeptide was purified on a nickel nitrilotriacetic acid (Ni-NTA) agarose column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the purity and molecular mass of iturin A was 41 kDa. The yield of recombinant iturin A was found to be 60 g/L with a 6.7-fold increase in comparison with our previously published study on the wild strain. The approach of cloning a functional fragment of partial iturin A resulted in the increased production of the lipopeptide. When motor oil was used, recombinant protein iturin A revealed a biosurfactant property with a 74 ± 1.9% emulsification index (E24). Purified recombinant protein iturin A was characterized by mass spectrometry. MALDI-TOF spectra of trypsin digestion (protein/trypsin of 50:1 and 25:1) showed desired digested mass peaks for the protein, further confirming the identity of iturin A. The iturin A structure was elucidated based on distinctive spectral bands in Raman spectra, which revealed the presence of a peptide backbone and lipid. Recombinant iturin A was employed for enhanced oil recovery through a sand-packed column that yielded 61.18 ± 0.85% additional oil. Hence, the novel approach of the high-level expression of iturin A (lipopeptide) as a promising biosurfactant employed for oil recovery from Bacillus aryabhattai is not much reported. Thus, recombinant iturin A demonstrated its promising ability for efficient oil recovery, finding specific applications in petroleum industries.

Enzymatic thioamidation of peptide backbones.

Thioamides are found in a few natural products and two known protein assemblies: the Escherichia coli ribosome and methyl-coenzyme M reductase (MCR) from methane-metabolizing archaea. Compared to an amide, thioamides alter the physical and chemical properties of peptide backbones, including the conformation dynamics, proteolytic stability, hydrogen-bonding capabilities, and possibly reactivity of a protein when installed. Recently, there has been significant progress in elucidating enzymatic post-translational thioamide installation, with most work leveraging the archaeal MCR-modifying enzymes. This chapter describes the protocols used for the in vitro enzymatic thioamidation of MCR-derived peptides, including polypeptide overexpression, purification, reaction reconstitution, and mass spectrometry-based product analysis. In addition, we highlight the protocols used for the biochemical, kinetics, and binding studies using recombinant enzymes obtained heterologously from E. coli. We anticipate that these methods will serve to guide future studies on peptide post-translational thioamidation, as well as other peptide backbone modifications using a parallel workflow.

Performance study of a sterilization box using a combination of heat and ultraviolet light irradiation for the prevention of COVID-19.

SARS-CoV-2 virus and other pathogenic microbes are transmitted to the environment through contacting surfaces, which need to be sterilized for the prevention of COVID-19 and related diseases. In this study, a prototype of a cost-effective sterilization box is developed to disinfect small items. The box utilizes ultra violet (UV) radiation with heat. For performance assessment, two studies were performed. First, IgG (glycoprotein, a model protein similar to that of spike glycoprotein of SARS-COV-2) was incubated under UV and heat sterilization. An incubation with UV at 70 °C for 15 min was found to be effective in unfolding and aggregation of the protein. At optimized condition, the hydrodynamic size of the protein increased to ~171 nm from ~5 nm of the native protein. Similarly, the OD280 values also increased from 0.17 to 0.78 indicating the exposure of more aromatic moieties and unfolding of the protein. The unfolding and aggregation of the protein were further confirmed by the intrinsic fluorescence measurement and FTIR studies, showing a 70% increase in the ß-sheets and a 22% decrease in the α-helixes of the protein. The designed box was effective in damaging the protein's native structure indicating the effective inactivation of the SARS-COV-2. Furthermore, the incubation at 70 °C for 15 min inside the chamber resulted in 100% antibacterial efficacy for the clinically relevant E.coli bacteria as well as for bacteria collected from daily use items. It is the first detailed performance study on the efficacy of using UV irradiation and heat together for disinfection from virus and bacteria.

Functional elucidation of TfuA in peptide backbone thioamidation.

YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser-Lys catalytic pair.

A Natural Quinazoline Derivative from Marine Sponge Hyrtios erectus Induces Apoptosis of Breast Cancer Cells via ROS Production and Intrinsic or Extrinsic Apoptosis Pathways.

Here, we report the therapeutic potential of a natural quinazoline derivative (2-chloro-6-phenyl-8H-quinazolino[4,3-b]quinazolin-8-one) isolated from marine sponge Hyrtios erectus against human breast cancer. The cytotoxicity of the compound was investigated on a human breast carcinoma cell line (MCF-7). Antiproliferative activity of the compound was estimated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assay showed significant inhibition of MCF-7 cells viability with the IC50 value of 13.04 ± 1.03 µg/mL after 48 h. The compound induced down-regulation of anti-apoptotic Bcl-2 protein and increase in the pro-apoptotic Bax/Bcl-2 ratio in MCF-7 cells. The compound activated the expression of Caspases-9 and stimulated downstream signal transducer Caspase-7. In addition, Caspase-8 showed remarkable up-regulation in MCF-7 cells treated with the compound. Moreover, the compound was found to promote oxidative stress in MCF-7 cells that led to cell death. In conclusion, the compound could induce apoptosis of breast carcinoma cells via a mechanism that involves ROS production and either extrinsic or intrinsic apoptosis pathways. The systemic toxic potential of the compound was evaluated in an in vivo mouse model, and it was found non-toxic to the major organs.

Reconstitution and Substrate Specificity of the Thioether-Forming Radical S-Adenosylmethionine Enzyme in Freyrasin Biosynthesis.

The radical non-α-carbon thioether peptides (ranthipeptides) are a newly described class of ribosomally synthesized and post-translationally modified peptide (RiPP). Ranthipeptide biosynthetic gene clusters are characterized by a Cys-rich precursor peptide and a radical S-adenosylmethionine (rSAM)-dependent enzyme that forms a thioether linkage between a Cys donor and an acceptor residue. Unlike the sulfur-to-α-carbon linked thioether peptides (sactipeptides), known ranthipeptides contain thioethers to either the ß- or γ-carbon (i.e., non-α-carbon) of an acceptor residue. Recently, we reported the discovery of freyrasin, a ranthipeptide from Paenibacillus polymyxa, which contains six thioethers from Cys-X3-Asp motifs present in the precursor peptide (PapA). The linkages are exclusively to the ß-carbon of Asp (S-Cß). In this report, we performed mutational analysis of PapA and the cognate thioether-forming rSAM enzyme (PapB) to define the substrate scope. Using a mass spectrometry-based activity assay, our data show that PapB is intolerant toward Ala and Asn in the acceptor position but tolerates Glu-containing variants. NMR spectroscopic data of a Glu variant demonstrated that the thioether linkage was to the 4-position of Glu (S-Cγ). Furthermore, we demonstrate that PapB is intolerant to expansion and contraction of the thioether motifs (Cys-Xn-Asp, n = 2 or 4), although a minimal substrate featuring only one Cys-X3-Asp motif was competent for thioether formation. Akin to the sactipeptides, PapB was dependent on a RiPP recognition element (RRE) to bind the cognate precursor peptide, with deletion resulting in loss-of-function in vivo. The activity of PapB could be restored in vivo by supplying the excised RRE in trans. Finally, we reconstituted the activity of PapB in vitro, which led to modification of all six Cys residues in PapA. These studies provide insights into ranthipeptide biosynthesis and expand our understanding of rSAM enzyme chemistry in natural product biosynthesis.

Mechanistic Basis for Ribosomal Peptide Backbone Modifications.

YcaO enzymes are known to catalyze the ATP-dependent formation of azoline heterocycles, thioamides, and (macro)lactamidines on peptide substrates. These enzymes are found in multiple biosynthetic pathways, including those for several different classes of ribosomally synthesized and post-translationally modified peptides (RiPPs). However, there are major knowledge gaps in the mechanistic and structural underpinnings that govern each of the known YcaO-mediated modifications. Here, we present the first structure of any YcaO enzyme bound to its peptide substrate in the active site, specifically that from Methanocaldococcus jannaschii which is involved in the thioamidation of the α-subunit of methyl-coenzyme M reductase (McrA). The structural data are leveraged to identify and test the residues involved in substrate binding and catalysis by site-directed mutagenesis. We also show that thioamide-forming YcaOs can carry out the cyclodehydration of a related peptide substrate, which underscores the mechanistic conservation across the YcaO family and allows for the extrapolation of mechanistic details to azoline-forming YcaOs involved in RiPP biosynthesis. A bioinformatic survey of all YcaOs highlights the diverse sequence space in azoline-forming YcaOs and suggests their early divergence from a common ancestor. The data presented within provide a detailed molecular framework for understanding this family of enzymes, which reconcile several decades of prior data on RiPP cyclodehydratases. These studies also provide the foundational knowledge to impact our mechanistic understanding of additional RiPP biosynthetic classes.

Antibacterial Strategy against H. pylori: Inhibition of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis.

Aminofutalosine synthase (MqnE) catalyzes an important rearrangement reaction in menaquinone biosynthesis by the futalosine pathway. In this Letter, we report the identification of previously unreported inhibitors of MqnE using a mechanism-guided approach. The best inhibitor shows efficient inhibitory activity against H. pylori (IC50 = 1.8 ± 0.4 µM) and identifies MqnE as a promising target for antibiotic development.

Menaquinone Biosynthesis: Biochemical and Structural Studies of Chorismate Dehydratase.

Menaquinone (MK, vitamin K) is a lipid-soluble quinone that participates in the bacterial electron transport chain. In mammalian cells, vitamin K functions as an essential vitamin for the activation of several proteins involved in blood clotting and bone metabolism. MqnA is the first enzyme on the futalosine-dependent pathway to menaquinone and catalyzes the aromatization of chorismate by water loss. Here we report biochemical and structural studies of MqnA. These studies suggest that the dehydration reaction proceeds by a variant of the E1cb mechanism in which deprotonation is slower than water loss and that the enol carboxylate of the substrate is serving as the base.

Biosynthesis and Chemical Applications of Thioamides.

Thioamidation as a posttranslational modification is exceptionally rare, with only a few reported natural products and exactly one known protein example (methyl-coenzyme M reductase from methane-metabolizing archaea). Recently, there has been significant progress in elucidating the biosynthesis and function of several thioamide-containing natural compounds. Separate developments in the chemical installation of thioamides into peptides and proteins have enabled cell biology and biophysical studies to advance the current understanding of natural thioamides. This review highlights the various strategies used by Nature to install thioamides in peptidic scaffolds and the potential functions of this rare but important modification. We also discuss synthetic methods used for the site-selective incorporation of thioamides into polypeptides with a brief discussion of the physicochemical implications. This account will serve as a foundation for the further study of thioamides in natural products and their various applications.

Novel enzymology in futalosine-dependent menaquinone biosynthesis.

The recently discovered futalosine-dependent menaquinone biosynthesis pathway employs radical chemistry for the naphthoquinol core assembly. Mechanistic studies on this pathway have resulted in the discovery of novel reaction motifs. MqnA is the first example of a chorismate dehydratase. MqnE is the first example of a radical SAM enzyme that catalyzes the addition of the 5'-deoxyadenosyl radical to the substrate double bond rather than hydrogen atom abstraction. Both MqnE and MqnC reaction sequences involve radical additions to a benzene ring followed by formation of an aryl radical anion intermediate. The enzymology of the tailoring reactions after dihydroxynaphthoic acid formation remains to be elucidated. Since the futalosine-dependent menaquinone biosynthesis pathway is absent in humans, mechanistic studies on this pathway may promote the development of new antibiotics.

Aminofutalosine Synthase (MqnE): A New Catalytic Motif in Radical SAM Enzymology.

Aminofutalosine synthase (MqnE) is a radical SAM enzyme involved in the futalosine-dependent menaquinone biosynthetic pathway. Its ability to add the 5'-deoxyadenosyl radical to the substrate-rather than abstract a hydrogen atom-and to catalyze radical addition to a stable benzene ring gives it a unique place in the radical SAM superfamily and required the development of new strategies for trapping radical intermediates. This chapter describes the methodologies used for enzyme overexpression, purification, and in vitro reconstitution. We also describe the development of fast, radical triggered, carbon-halogen bond fragmentation reactions for the trapping of intermediates. We anticipate that these methods will be of general use in the study of other transient enzymatic radicals.

Measurement of Kinetics and Active Site Distances in Metalloenzymes Using Paramagnetic NMR with 13C Hyperpolarization.

Paramagnetic relaxation enhancement (PRE) conjoint with hyperpolarized NMR reveals structural information on the enzyme-product complex in an ongoing metalloenzyme-catalyzed reaction. Substrates of pseudouridine monophosphate glycosidase are hyperpolarized using the dynamic nuclear polarization (DNP) method. Time series of 13C NMR spectra are subsequently measured with the enzyme containing diamagnetic Mg2+ or paramagnetic Mn2+ ions in the active site. The differences of the signal evolution and line widths in the Mg2+ vs Mn2+ reactions are explained through PRE in the enzyme-bound product, which is in fast exchange with its free form. Here, a strong distance dependence of the paramagnetically enhanced relaxation rates enables the calculation of distances from product atoms to the metal center in the complexed structure. The same method can be used to add structural information to real-time characterizations of chemical processes involving compounds with naturally present or artificially introduced paramagnetic sites.

Enzymatic reconstitution of ribosomal peptide backbone thioamidation.

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.

Mechanism of a Class C Radical S-Adenosyl-l-methionine Thiazole Methyl Transferase.

The past decade has seen the discovery of four different classes of radical S-adenosylmethionine (rSAM) methyltransferases that methylate unactivated carbon centers. Whereas the mechanism of class A is well understood, the molecular details of methylation by classes B-D are not. In this study, we present detailed mechanistic investigations of the class C rSAM methyltransferase TbtI involved in the biosynthesis of the potent thiopeptide antibiotic thiomuracin. TbtI C-methylates a Cys-derived thiazole during posttranslational maturation. Product analysis demonstrates that two SAM molecules are required for methylation and that one SAM (SAM1) is converted to 5'-deoxyadenosine and the second SAM (SAM2) is converted to S-adenosyl-l-homocysteine (SAH). Isotope labeling studies show that a hydrogen is transferred from the methyl group of SAM2 to the 5'-deoxyadenosine of SAM1 and the other two hydrogens of the methyl group of SAM2 appear in the methylated product. In addition, a hydrogen appears to be transferred from the ß-position of the thiazole to the methyl group in the product. We also show that the methyl protons in the product can exchange with solvent. A mechanism consistent with these observations is presented that differs from other characterized radical SAM methyltransferases.