Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA) by combination of oxacillin and a bioactive compound from Ramalinaroesleri.

Lichens are known to be useful and important in ethanopharmacology since ages and still possess substantial interest in alternative medical practices around the world. The intent of this investigation was to evaluate and to understand the antibacterial potential of usnic acid which was isolated from Himalyan fruticose lichen Ramalina roesleri. Usnic acid is predicted for its pharmaceutical properties through in -silico studies. Binding efficiency of usnic acid with Penicillin binding protein-PBP2a, a protein which is responsible for conferring resistance in Staphylococcus aureus was accessed using in-silico interaction assays comparing with oxacillin and ceftaroline. Further, the validation of in-silico modelling was checked by determining the antibacterial potential of usnic acid against methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates. In total, 28 clinical isolates collected from hospitals/medical students were included in the study and the anti-Staphylococcal activity was determined using agar plate dilution method followed by time-kill kinetics and synergistic studies. The scanning electron microscopic (SEM) pictures were obtained to show the cell wall disruption of MRSA by usnic acid. Docking results clearly indicated the enhanced binding potential of usnic acid (Glide XP G Score: 10.968; Glide energy -64.869) with PBP2a which is better than the energy range of reference compound, oxacillin (Glide XP G Score: 6.596; Glide energy -53.285) and roughly comparable to the co-crystallized ligand ceftaroline (Glide XP G Score: 12.20; Glide energy -70.322). Cefteroline is known to be more active against MRSA compared to oxacillin. The minimum inhibitory concentrations (MICs) of usnic acid against the clinical isolates of MRSA and reference strain (NCTC-6571) were in the range of 32-128 µg/ml. The high affinity of usnic acid to bind with PBP2a which is demonstrated via in-silico studies is further confirmed by the impressive inhibitory activity of usnic acid on MRSA clinical isolates.

Comparison of protein acetyltransferase action of CRTAase with the prototypes of HAT.

Our laboratory is credited for the discovery of enzymatic acetylation of protein, a phenomenon unknown till we identified an enzyme termed acetoxy drug: protein transacetylase (TAase), catalyzing the transfer of acetyl group from polyphenolic acetates to receptor proteins (RP). Later, TAase was identified as calreticulin (CR), an endoplasmic reticulum luminal protein. CR was termed calreticulin transacetylase (CRTAase). Our persistent study revealed that CR like other families of histone acetyltransferases (HATs) such as p300, Rtt109, PCAF, and ESA1, undergoes autoacetylation. The autoacetylated CR was characterized as a stable intermediate in CRTAase catalyzed protein acetylation, and similar was the case with ESA1. The autoacetylation of CR like that of HATs was found to enhance protein-protein interaction. CR like HAT-1, CBP, and p300 mediated the acylation of RP utilizing acetyl CoA and propionyl CoA as the substrates. The similarities between CRTAase and HATs in mediating protein acylation are highlighted in this review.

Characterization of 7-amino-4-methylcoumarin as an effective antitubercular agent: structure-activity relationships.

OBJECTIVES: The objective of the present study was to evaluate the antitubercular activity of amino and acyl amino derivatives of coumarins when used alone and in combination with isoniazid, rifampicin, streptomycin or ethambutol, and to decipher the mode of action of the most effective agent. METHODS: A series of amino and acyl amino coumarins were synthesized and screened for activity against the Mycobacterium tuberculosis H37Rv strain. These compounds were further evaluated by standard assay procedures to determine their MBCs, fractional inhibitory concentration index values and cytotoxicities. The MICs for a susceptible and a multidrug-resistant clinical isolate of M. tuberculosis were also determined. Electron and fluorescence microscopy of the test compound-treated mycobacterial samples were also carried out in an attempt to find out the target of action. RESULTS: 7-Amino-4-methylcoumarin (7-amino-4-methyl-2H-chromen-2-one; NA5) displayed the lowest MIC of 1 mg/L against not only H37Rv but also the susceptible as well as the multidrug-resistant clinical isolates. Certain acyl amino coumarins were also found to inhibit the aforementioned strains and isolates with MICs in the range of 1.0-3.5 mg/L. They were also found to act in synergy with isoniazid/rifampicin. Electron microscopy revealed the cell-wall-attacking characteristic of these compounds, while fluorescence microscopy indicated that mycolic acid might be the target of action. CONCLUSIONS: The present study clearly demonstrated the in vitro antitubercular potential of the novel drug candidate NA5. Further studies are warranted to establish the in vivo efficacy and therapeutic potential of NA5.

Single nucleotide polymorphism in the genes of mce1 and mce4 operons of Mycobacterium tuberculosis: analysis of clinical isolates and standard reference strains.

BACKGROUND: The presence of four mammalian cell entry (mce) operons in Mycobacterium tuberculosis suggests the essentiality of the functions of the genes in these operons. The differential expression of the four mce operons in different phases of in vitro growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome.Here we investigate the extent of polymorphism in eight genes in the mce1 and mce4 operons of M. tuberculosis from four standard reference strains (H37Rv, H37Ra, LVS (Low Virulent Strain) and BCG) and 112 clinical isolates varying in their drug susceptibility profile, analysed by direct sequencing and Sequenom MassARRAY platform. RESULTS: We discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of mce1 and mce4 operons revealed that yrbE1A [Rv0167] was most polymorphic in mce1 operon while yrbE4A [Rv3501c] and lprN [Rv3495c] had the highest number of SNPs in the mce4 operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to M. tuberculosis using web servers PolyPhen and PMut, which predicted five deleterious nonsynonymous SNPs. A mutation from proline to serine at position 359 of the native Mce1A protein was most deleterious as predicted by both PolyPhen and PMut servers. Energy minimization of the structure of native Mce1A protein and mutated protein was performed using InsightII. The mutated Mce1A protein showed structural changes that could account for the effects of this mutation. CONCLUSIONS: Our results show that SNPs in the coding sequences of mce1 and mce4 operons in clinical isolates can be significantly high. Moreover, mce4 operon is significantly more polymorphic than mce1 operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in mce1 operon and synonymous substitutions are more in mce4 operon. In silico modeling predict that nonsynonymous SNP at mce1A [Rv0169], a virulence gene could play a pivotal role in causing functional changes in M. tuberculosis that may reflect upon the biology of the bacteria.

Site-directed mutagenesis in the P-domain of calreticulin transacylase identifies Lys-207 as the active site residue.

In silico-docking studies from previous work have suggested that Lys-206 and lys-207 of calreticulin (CR) play a pivotal key role in its well-established transacetylation activity. To experimentally validate this prediction, we introduced three mutations at lysine residues of P-domain of CR: K → A, P mut-1 (K -206, -209), P mut-2 (K -206, -207) and P mut-3 (K -207, -209) and analyzed their immunoreactivity and acetylation potential. The clones of wild-type P-domain (P wt ) and three mutated P-domain (P mut-1, P mut-2 and P mut-3) were expressed in pTrcHis C vector and the recombinant P wt , P mut-1 , P mut-2 and P mut-3 proteins were purified by Ni-NTA affinity chromatography. Screening of the transacylase activity (TAase) by the Glutathione S Transferase (GST) assay revealed that the TAase activity was associated with the P wt and P mut-1 while P mut-2 and P mut-3 did not show any activity. The immune-reactivity to anti-lysine antibody was also retained only by the P mut-1 in which the Lys-207 was intact. Retention of the TAase activity and immunoreactivity of P mut-1 with mutations introduced at Lys-206, Lys-209, while its loss with a mutation at Lys-207 residue indicated that lysine-207 of P-domain constitutes the active site residue controlling TAase activity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02659-1.

Hybridization of Fluoro-amodiaquine (FAQ) with Pyrimidines: Synthesis and Antimalarial Efficacy of FAQ-Pyrimidines.

To evade the possible toxicity associated with the formation of quinone-imine metabolite in amodiaquine (AQ), the para-hydroxyl group was replaced with a -F atom, and the resulting 4'-fluoro-amodiaquine (FAQ) was hybridized with substituted pyrimidines. The synthesized FAQ-pyrimidines displayed better in vitro potency than chloroquine (CQ) against the resistant P. falciparum strain (Dd2), exhibiting up to 47.3-fold better activity (IC50: 4.69 nM) than CQ (IC50: 222 nM) and 2.8-fold better potency than artesunate (IC50: 13.0 nM). Twelve compounds exhibited better antiplasmodial activity than CQ against the CQ-sensitive (NF54) strain. Two compounds were evaluated in vivo against a P. berghei-mouse malaria model. Mechanistic heme-binding studies, computational docking studies against Pf-DHFR and in vitro microsomal stability studies were performed for the representative molecules of the series to assess their antimalarial efficacy.

Establishment of glutamine synthetase of Mycobacterium smegmatis as a protein acetyltransferase utilizing polyphenolic acetates as the acetyl group donors.

Acetoxy Drug: Protein Transacetylase (TAase) mediating the transfer of acetyl group(s) from polyphenolic acetates (PA) to certain functional proteins in mammalian cells was identified by our earlier investigations. TAase activity was characterized in the cell lysates of Mycobacterium smegmatis and the purified protein was found to have M(r) 58,000. TAase catalysed protein acetylation by a model acetoxy drug 7,8-diacetoxy-4-methylcoumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with an anti-acetyllysine antibody. The specificity of the TAase of M. smegmatis (MTAase) to various acetoxycoumarins was found to be in the order DAMC > 7-AMC > 6-AMC > 4-AC > 3-AC > ABP. Also, the N-terminal sequence of purified MTAase was found to perfectly match with glutamine synthetase (GS) of M. smegmatis. The identity of MTAase with GS was confirmed by the observation that the purified MTAase as well as the purified recombinant GS exhibited all the properties of GS. The finding that purified Escherichia coli GS was found to have substantial TAase activity highlighted the TAase function of GS in other bacteria. These results conclusively established for the first time the protein acetyltransferase function of GS of M. smegmatis.

Calreticulin transacetylase mediates the acetylation of nitric oxide synthase by polyphenolic acetate.

Our earlier investigations identified acetoxy drug: protein transacetylase (TAase), a unique enzyme in the endoplasmic reticulum (ER) catalyzing the transfer of acetyl groups from polyphenolic acetates (PA) to certain functional proteins. Recently we have established the identity of TAase with ER protein calreticulin (CR) and subsequently transacetylase function of CR was termed calreticulin transacetylase (CRTAase). CRTAase was purified and characterized from human placenta. CRTAase catalyzed the acetylation of a receptor protein nNOS, by a model PA 7, 8-diacetoxy-4-methylcoumarin (DAMC), which was visually confirmed by using antiacetyl lysine. The aim of this report was to provide tacit proof by providing mass spectrometry evidence for CRTAase catalyzed acetylation of purified nNOS by DAMC. For this purpose, purified nNOS was incubated with DAMC and CRTAase, the modified nNOS was analyzed by nanoscale LC-MS/MS, which recorded 11 distinct peptides with a significant score as acetylated on lysine residues. The distribution was in order: lysines-24, -33, -38, -131, and -229 of the PDZ domain, Lys-245 of the oxygenase domain, Lys-754 and -856 of FMN binding domain, Lys-989 of connecting domain and Lys-1300, -1321, and -1371 of the NADPH-binding domain were acetylated. The results documented in this paper highlighted for the first time modification of nNOS by way of acetylation. Our earlier work recorded the profound activation of platelet NADPH cytochrome P-450 reductase and the acetylation of the reductase protein by DAMC, which also remarkably enhanced intracellular levels of nitric oxide. The results reported here coupled with the aforementioned previous observations strongly implicate the possible role of the acetylation of the reductase domain of nitric oxide synthase (NOS) in the NOS activation. In addition, the acetylation of nNOS can be expected to potentiate the interaction with CR, eventually leading to the augmented catalytic activity of NOS and expression of the related biological effects.

Synthesis and antimycobacterial activity of 1-(ß-d-Ribofuranosyl)-4-coumarinyloxymethyl- / -coumarinyl-1,2,3-triazole.

A series of ß-d-ribofuranosyl coumarinyl-1,2,3-triazoles have been synthesized by Cu-catalyzed cycloaddition reaction between azidosugar and 7-O-/7-alkynylated coumarins in 62-70% overall yields. The in vitro antimycobacterial activity evaluation of the synthesized triazolo-conjugates against Mycobacterium tuberculosis revealed that compounds were bactericidal in nature and some of them were found to be more active than one of the first line antimycobacterial drug ethambutol against sensitive reference strain H37Rv, and 7 to 420 times more active than all four first line antimycobacterial drugs (isoniazid, rifampicin, ethambutol and streptomycin) against multidrug resistant clinical isolate 591. Study of in silico pharmacokinetic profile indicated the drug like characters for the test molecules. Further, transmission electron microscopic experiments revealed that these compounds interfere with the constitution of bacterial cell wall possibly by targeting mycobacterial InhA and DNA gyrase enzymes. Study conducted on the activities of the test compounds on bacterial InhA and DNA gyrase revealed that the most bactericidal test compound, N1-(ß-d-ribofuranosyl)-C4-(4-methylcoumarin-7-oxymethyl)-1,2,3-triazole (6b) and its corresponding directly linked conjugate N1-(ß-d-ribofuranosyl)-C4-(4-methylcoumarin-7-yl)-1,2,3-triazole (11b) significantly inhibited the activity of both the enzymes. The results were further supported by molecular docking studies of the compound 6b and 11b with bacterial InhA and DNA gyrase B enzymes. Further, the cytotoxicity study of some of the better active compounds on THP-1 macrophage cell line using MTT assay showed that the synthesized compounds were non-cytotoxic.

Ellagic acid peracetate is superior to ellagic acid in the prevention of genotoxicity due to aflatoxin B1 in bone marrow and lung cells.

Earlier observations carried out in our laboratory highlighted the mode of action of acetoxy 4-methylcoumarins and quercetin pentaacetate in preventing the genotoxicity of aflatoxin B1 (AFB1). We have extended the observation to an acetoxy biscoumarin i.e. ellagic acid peracetate (EAPA), which unlike ellagic acid (EA) has demonstrated time-dependent inhibition of liver microsomes catalysed AFB1-epoxidation as measured by AFB1 binding to DNA. EAPA was more potent than EA in preventing bone marrow and lung cells from AFB1-induced genotoxicity. EAPA was acted upon by microsomal acetoxy drug:protein transacetylase (TAase) leading to modulation of the catalytic activity of certain functional proteins (cytochrome P450, NADPH cytochrome c reductase and glutathione S-transferase), possibly by way of protein acetylation.

Design, synthesis and evaluation of 4-aminoquinoline-purine hybrids as potential antiplasmodial agents.

A novel series of 4-aminoquinoline-purine hybrids were synthesized and assessed for their antiplasmodial activity against CQ-sensitive and CQ-resistant strains of P. falciparum. It was envisaged that linking of the 4-aminoquinoline pharmacophore (targeting heme-detoxification pathway of malarial parasite) with the purine functionality (targeting plasmodial HG(X)PRT enzyme) will produce a hybrid antiplasmodial agent with increased potency. The synthesized hybrids displayed good antiplasmodial activities against both the sensitive and resistant strains of P. falciparum with up to six-fold better activity (compound 10i, IC50: 0.08 µM) compared to the reference drug CQ (IC50: 0.5 µM) against the resistant strain. The synthesized compounds were also checked for their cytotoxicity towards mammalian cells and with the exception of two compounds out of the twenty synthesized hybrids, all others were non-cytotoxic up to 11.86 µM concentration. Mechanistic heme-binding studies were performed to identify the mechanism of action of the synthesized molecules and good binding interactions were observed. Computational docking studies showed that the most active hybrids dock well within the binding site of HGPRT protein. In silico ADME predictions of the most active hybrids showed that these compounds possess good pharmacokinetic behavior.

Investigations on Binding Pattern of Kinase Inhibitors with PPARγ: Molecular Docking, Molecular Dynamic Simulations, and Free Energy Calculation Studies.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a potential target for the treatment of several disorders. In view of several FDA approved kinase inhibitors, in the current study, we have investigated the interaction of selected kinase inhibitors with PPARγ using computational modeling, docking, and molecular dynamics simulations (MDS). The docked conformations and MDS studies suggest that the selected KIs interact with PPARγ in the ligand binding domain (LBD) with high positive predictive values. Hence, we have for the first time shown the plausible binding of KIs in the PPARγ ligand binding site. The results obtained from these in silico investigations warrant further evaluation of kinase inhibitors as PPARγ ligands in vitro and in vivo.

Corrigendum to The Competence of 7,8-Diacetoxy-4-Methylcoumarin and Other Polyphenolic Acetates in Mitigating the Oxidative Stress and their Role in Angiogenesis.

In the Original Research Article entitled "The Competence of 7, 8-Diacetoxy-4-methylcoumarin and other Polyphenolic Acetates in Mitigating the Oxidative Stress and their Role in Angiogenesis" Published in Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 2, on page no. 179, the order of author names was rearranged because second authorship is acceptable as they only acknowledge the first and the second authorship as per the new policies of Medical Council of India. The order of authors should be read as follows: Rini Joshi, Vishwajeet Rohil, Shvetambri Arora, Sushma Manral, Ajit Kumar, Sanjay Goel, Nivedita Priya, Prabhjoth Singh, Prija Ponnan, Suvro Chatterji, Bilikere S. Dwarakanath, Daman Saluja, Diwan S. Rawat, Ashok K. Prasad, Luciano Saso, Ekta Kohli, Anthony L. DePass, Marc E. Bracke, Virinder S. Parmar and Hanumantharao G. Raj.

Anti-methicillin resistant Staphylococcus aureus activity, synergism with oxacillin and molecular docking studies of metronidazole-triazole hybrids.

MRSA causes 60-70% of Staphylococcus aureus infection in hospitals and it has developed resistance against the currently available drugs. Interestingly, a series of 35 metronidazole-triazole hybrids on screening against MRSA were found to be active. Compound 22 was found to be effective at 4 µg/mL concentration against nine strains of MRSA. The inhibitory activity was further enhanced upto 1 µg/mL when this compound was used in combination with oxacillin in 1:1 ratio. All the compounds were found to be non-toxic in THP-1 cell line upto a concentration of 50 µM. The time-kill kinetics studies suggested bacteriostatic nature of the compounds. In silico studies show that these compounds interact with Thr600, Ser598, Asn464, His583 and Tyr446 in the active site of PBP2a crystal structure from MRSA.

4-Aminoquinoline-pyrimidine hybrids: synthesis, antimalarial activity, heme binding and docking studies.

A series of novel 4-aminoquinoline-pyrimidine hybrids has been synthesized and evaluated for their antimalarial activity. Several compounds showed promising in vitro antimalarial activity against both CQ-sensitive and CQ-resistant strains with high selectivity index. All the compounds were found to be non-toxic to the mammalian cell lines. Selected compound 7g exhibited significant suppression of parasitemia in the in vivo assay. The heme binding studies were conducted to determine the mode of action of these hybrid molecules. These compounds form a stable 1:1 complex with hematin suggesting that heme may be one of the possible targets of these hybrids. The interaction of these conjugate hybrids was also investigated by the molecular docking studies in the binding site of PfDHFR. The pharmacokinetic property analysis of best active compounds was also studied using ADMET prediction.

The Competence of 7,8-Diacetoxy-4-Methylcoumarin and Other Polyphenolic Acetates in Mitigating the Oxidative Stress and their Role in Angiogenesis.

The potential role of polyphenolic acetate (PA) in causing diverse biological and pharmacological actions has been well studied in our laboratory. Our investigations, for the first time, established the role of calreticulin transacetylase (CRTAase) in catalyzing the acetylation of nitric oxide synthase (NOS) by Pas leading to robust activation of NOS. 7, 8- Diacetoxy-4-methylcoumarin (DAMC) and other acetoxycoumarins augmented the expression of thioredoxin (TRX) and vascular endothelial growth factor (VEGF) in human peripheral blood mononuclear cells (PBMCs). These findings substantiated our earlier observations that DAMC was a superb inducer of angiogenesis. The enhanced expression of thioredoxin reductase (TRXR) and diminished expression of thioredoxin interacting protein (TRXIP) leading to increased expression and activity of TRX in PBMCs due to the action of DAMC was revealed by real time RT-PCR analysis. The possible activation of TRX due to acetylation was confirmed by the fact that TRX activity of PBMCs was enhanced by various acetoxycoumarins in tune with their affinities to CRTAase as substrates. DAMC caused enhanced production of NO by way of acetylation of NOS as mentioned above and thereby acted as an inducer of VEGF. Real time RT-PCR and VEGF ELISA results also revealed the overexpression of TRX. DAMC and other PAs were found to reduce the oxidative stress in cells as proved by significant reduction of intracellular ROS levels. Thus, the crucial role of TRX in DAMC-induced angiogenesis with the involvement of VEGF was established.

The Competence Of 7,8-Diacetoxy-4-Methylcoumarinand Other Polyphenolic Acetates In Mitigating The Oxidative Stress And Their Role In Angiogenesis.

The potential role of polyphenolic acetate (PA) in causing diverse biological and pharmacological actions has been well studied in our laboratory. Our investigations, for the first time, established the role of calreticulin transacetylase (CRTAase) in catalyzing the acetylation of nitric oxide synthase (NOS) by Pas leading to robust activation of NOS. 7, 8-Diacetoxy-4-methylcoumarin (DAMC) and other acetoxycoumarins augmented the expression of thioredoxin (TRX) and vascular endothelial growth factor (VEGF) in human peripheral blood mononuclear cells (PBMCs). These findings substantiated our earlier observations that DAMC was a superb inducer of angiogenesis. The enhanced expression of thioredoxin reductase (TRXR) and diminished expression of thioredoxin interacting protein (TRXIP) leading to increased expression and activity of TRX in PBMCs due to the action of DAMC was revealed by real time RT-PCR analysis. The possible activation of TRX due to acetylation was confirmed by the fact that TRX activity of PBMCs was enhanced by variousacetoxycoumarins in tune with their affinities to CRTAase as substrates. DAMC caused enhanced production of NO by way of acetylation of NOS as mentioned above and thereby acted as an inducer of VEGF. Real time RT-PCR and VEGF ELISA results also revealed the overexpression of TRX. DAMC and other PAs were found to reduce the oxidative stress in cells as proved by significant reduction of intracellular ROS levels. Thus, the crucial role of TRX in DAMC-induced angiogenesis with the involvement of VEGF was established.

Comparative specificities of Calreticulin Transacetylase to O-acetyl, N-acetyl and S-acetyl derivative of 4-methylcoumarins and their inhibitory effect on AFB1-induced genotoxicity in vitro and in vivo.

We have earlier conclusively established the Calreticulin Transacetylase (CRTAase) catalyzed modifications of functional proteins such as cytochrome-P450-linked mixed function oxidases (Cyt-P450-linked MFOs), NADPH cytochrome c reductase, and glutathione S-transferase by acetoxy derivatives of polyphenols. In this study, we have investigated the comparative specificities of CRTAase to N-acetyl derivative, 7-acetamido-4-methylcoumarin (7-N-AMC), O-acetyl derivative, 7-acetoxy-4-methylcoumarin (7-AMC), S-acetyl derivative, 7-thioacetyl-4-methycoumarin (7-S-AMC) and their parent compounds in the modulation of catalytic activities of aforesaid proteins. Special attention concentrated on the comparative inhibitory effect of aforesaid acetyl moiety on Cyt-P450-linked MFOs such as 7-ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-dealkylase (PROD) and aflatoxin B(1) (AFB(1))-induced genotoxicity in vitro and in vivo. The results clearly indicated that N-acetyl and O-acetyl derivatives were better substrates for CRTAase while the S-acetyl was found to be a poorer substrate. Our study involving atomic charge, charge density and molecular electrostatic potential (MEP) calculations indicated the pivotal role of electronegativity and charge distribution values of O, N and S atoms of the acetyl group at C-7 position of the 4-methylcoumarins in CRTAase activity. These facts reinforce our hypothesis that the CRTAase catalyzed modifications of the catalytic activities of aforesaid proteins by acetyl derivative of 4-methylcoumarins is probably due to acetylation of these proteins.

Protein acyltransferase function of purified calreticulin: the exclusive role of P-domain in mediating protein acylation utilizing acyloxycoumarins and acetyl CoA as the acyl group donors.

The distinct biochemical function of endoplasmic reticulum (ER) protein Calreticulin (CR) catalyzing the transfer of acyl group from acyloxycoumarin to a receptor protein was termed calreticulin transacylase (CRTAase). The present study, unlike the previous reports of others utilizing CR-deficient cells alone, dealt with the recombinant CR domains of Heamonchus contortus (rhCRTAase) in order to examine their CRTAase activity. P-domain of rhCR unlike N- and C-domains was found to be endowed with CRTAase function. We have also observed for the first time acetyl CoA, as a substrate for rhCRTAase/P-domain mediated acetylation of recombinant Schistosoma japonicum glutathione S-transferase (rGST). rhCRTAase/P-domain were also found to undergo autoacylation by acyloxycoumarins. Also, the isolated autoacylated rhCRTAase/P-domain in non-denatured form alone exhibited the ability to transfer acyl group to rGST indicating the stable intermediate nature of acylated CR. P-domain catalyzed acetylation of rGST by 7,8-Diacetoxy-4-methylcoumarin or acetyl CoA resulted in the modification of several lysine residues in common was evidenced by LC-MS/MS analysis. The putative site of the binding of acyloxycoumarins with CR was predicted by computational blind docking studies. The results showed the involvement of two lysine residues Lys-173 and Lys-174 present in P-domain for binding acyloxycoumarins and acetyl CoA thus highlighting that the active site for the CRTAase activity would reside in the P-domain of CR. Certain ER proteins are known to undergo acetylation under the physiological conditions involving acetyl CoA. These results demonstrating CRTAase mediated protein acetylation by acetyl CoA may hint at CR as the possible protein acetyltransferase of the ER lumen.