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.

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.

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.

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.

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.

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.

Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) display a diverse range of structures and continue to expand as a natural product class. Accordingly, RiPPs exhibit a wide array of bioactivities, acting as broad and narrow spectrum growth suppressors, antidiabetics, and antinociception and anticancer agents. Because of these properties, and the complex repertoire of post-translational modifications (PTMs) that give rise to these molecules, RiPP biosynthesis has been intensely studied. RiPP biosynthesis often involves enzymes that perform unique chemistry with intriguing reaction mechanisms, which attract chemists and biochemists alike to study and re-engineer these pathways. One particular type of RiPP biosynthetic enzyme is the so-called radical S-adenosylmethionine (rSAM) enzyme, which utilizes radical-based chemistry to install several distinct PTMs. Here, we describe the rSAM enzymes characterized over the past decade that catalyze six reaction types from several RiPP biosynthetic pathways. We present the current state of mechanistic understanding and conclude with possible directions for future characterization of this enzyme family.

Aminofutalosine Synthase: Evidence for Captodative and Aryl Radical Intermediates Using ß-Scission and SRN1 Trapping Reactions.

Aminofutalosine synthase (MqnE) is a radical SAM enzyme involved in the menaquinone biosynthetic pathway. In this communication, we propose a novel mechanism for this reaction involving the addition of the adenosyl radical to the substrate double bond to form a captodative radical followed by rearrangement and decarboxylation to form an aryl radical anion which is then oxidized by the [4Fe-4S]+2 cluster. Consistent with this proposal, we describe the trapping of the captodative radical and the aryl radical anion using radical triggered C-Br fragmentation reactions. We also describe the trapping of the captodative radical by replacing the vinylic carboxylic acid with an amide.

Reconstitution and Substrate Specificity of the Radical S-Adenosyl-methionine Thiazole C-Methyltransferase in Thiomuracin Biosynthesis.

Thiomuracin is a thiopeptide antibiotic with potent activity toward Gram-positive drug-resistant bacteria. Thiomuracin is biosynthesized from a precursor peptide, TbtA, by a complex array of posttranslational modifications. One of several intriguing transformations is the C-methylation of thiazole, occurring at an unactivated sp2 carbon. Herein, we report the in vitro reconstitution of TbtI, the responsible radical S-adenosyl-methionine (rSAM) C-methyltransferase, which catalyzes the formation of 5-methylthiazole at a single site. Our studies demonstrate that a linear hexazole-bearing intermediate of TbtA is a substrate for TbtI whereas macrocyclized thiomuracin GZ is not. In determining the minimal substrate for TbtI, we found that the enzyme is functional when most of the leader peptide has been removed. The in vitro reconstitution of TbtI, a class C rSAM methyltransferase, further adds to the chemical versatility of rSAM enzymes, and informs on the complexity of thiomuracin biosynthesis.

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.

In Vitro Biosynthetic Studies of Bottromycin Expand the Enzymatic Capabilities of the YcaO Superfamily.

The bottromycins belong to the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Bottromycins exhibit unique structural features, including a hallmark macrolactamidine ring and thiazole heterocycle for which divergent members of the YcaO superfamily have been biosynthetically implicated. Here we report the in vitro reconstitution of two YcaO proteins, BmbD and BmbE, responsible for the ATP-dependent cyclodehydration reactions that yield thiazoline- and macrolactamidine-functionalized products, respectively. We also establish the substrate tolerance for BmbD and BmbE and systematically dissect the role of the follower peptide, which we show serves a purpose similar to canonical leader peptides in directing the biosynthetic enzymes to the substrate. Lastly, we leverage the expanded capabilities of YcaO proteins to conduct an extensive bioinformatic survey to classify known YcaO chemistry. This analysis predicts new functions remain to be uncovered within the superfamily.

Radical S-adenosylmethionine (SAM) enzymes in cofactor biosynthesis: a treasure trove of complex organic radical rearrangement reactions.

In this minireview, we describe the radical S-adenosylmethionine enzymes involved in the biosynthesis of thiamin, menaquinone, molybdopterin, coenzyme F420, and heme. Our focus is on the remarkably complex organic rearrangements involved, many of which have no precedent in organic or biological chemistry.

Biosynthetic Timing and Substrate Specificity for the Thiopeptide Thiomuracin.

The biosynthesis of the thiopeptide thiomuracin is a well-orchestrated process involving a multitude of posttranslational modifications. We show that six Cys residues of a precursor peptide are first cyclodehydrated and oxidized to thiazoles in an ordered, but nonlinear fashion that is leader-peptide-dependent. Then four alcohols are glutamylated and converted to alkenes in a C-to-N terminal directional process that is leader-peptide-independent. Finally, two of these alkenes undergo a formal [4 + 2] cycloaddition to form a trithiazole-substituted pyridine macrocycle. We describe here the factors that govern the substrate specificity and order of biosynthetic events that turn a ribosomal peptide into a powerful antibiotic.

Molecular expression, purification and structural characterization of recombinant L-Glutaminase from Streptomyces roseolus.

The present research reports the anti-cancer potential of recombinant L-Glutaminase from Streptomyces roseolus. L-Glutaminase gene was synthesized by codon-optimization, cloned and successfully expressed in E. coli BL21 (DE3). Affinity purified recombinant L-Glutaminase revealed a molecular mass of 32 kDa. Purified recombinant L-Glutaminase revealed stability at pH 7.0-8.0 with optimum activity at 70 °C further indicating its thermostable nature based on thermodynamic characterization. Recombinant L-Glutaminase exhibited profound stability in the presence of several biochemical parameters and demonstrated its metalloenzyme nature and was also found to be highly specific towards favorable substrate (l-Glutamine) based on kinetics. It demonstrated antioxidant property and pronounced cytotoxic effect against breast cancer (MCF-7 cell lines) in a dose dependent behavior with IC50 of 40.68 µg/mL. Matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis of desired mass peaks ascertained the recombinant L-Glutaminase identity. N-terminal amino acid sequence characterization through Edman degradation revealed highest resemblance for L-glutaminase within the Streptomyces sp. family. The purified protein was characterized structurally and functionally by employing spectroscopic methods like Raman, circular dichroism and nuclear magnetic resonance. The thermostability was assessed by thermogravimetric analysis. The outcomes of the study, suggests the promising application of recombinant L-Glutaminase as targeted therapeutic candidate for breast cancer.

Menaquinone biosynthesis: formation of aminofutalosine requires a unique radical SAM enzyme.

Menaquinone (MK, vitamin K2) is a lipid-soluble molecule that participates in the bacterial electron transport chain. In mammalian cells, MK functions as an essential vitamin for the activation of various proteins involved in blood clotting and bone metabolism. Recently, a new pathway for the biosynthesis of this cofactor was discovered in Streptomyces coelicolor A3(2) in which chorismate is converted to aminofutalosine in a reaction catalyzed by MqnA and an unidentified enzyme. Here, we reconstitute the biosynthesis of aminofutalosine and demonstrate that the missing enzyme (aminofutalosine synthase, MqnE) is a radical SAM enzyme that catalyzes the addition of the adenosyl radical to the double bond of 3-[(1-carboxyvinyl)oxy]benzoic acid. This is a new reaction type in the radical SAM superfamily.

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.

Pseudouridine monophosphate glycosidase: a new glycosidase mechanism.

Pseudouridine (Ψ), the most abundant modification in RNA, is synthesized in situ using Ψ synthase. Recently, a pathway for the degradation of Ψ was described [Preumont, A., Snoussi, K., Stroobant, V., Collet, J. F., and Van Schaftingen, E. (2008) J. Biol. Chem. 283, 25238-25246]. In this pathway, Ψ is first converted to Ψ 5'-monophosphate (ΨMP) by Ψ kinase and then ΨMP is degraded by ΨMP glycosidase to uracil and ribose 5-phosphate. ΨMP glycosidase is the first example of a mechanistically characterized enzyme that cleaves a C-C glycosidic bond. Here we report X-ray crystal structures of Escherichia coli ΨMP glycosidase and a complex of the K166A mutant with ΨMP. We also report the structures of a ring-opened ribose 5-phosphate adduct and a ring-opened ribose ΨMP adduct. These structures provide four snapshots along the reaction coordinate. The structural studies suggested that the reaction utilizes a Lys166 adduct during catalysis. Biochemical and mass spectrometry data further confirmed the existence of a lysine adduct. We used site-directed mutagenesis combined with kinetic analysis to identify roles for specific active site residues. Together, these data suggest that ΨMP glycosidase catalyzes the cleavage of the C-C glycosidic bond through a novel ribose ring-opening mechanism.

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.