The increase in the use of bactericides is a matter of grave concern and a serious threat to human health. The present situation demands rapid and efficient detection and elimination of antibiotic-resistant microbes. Herein, we report the synthesis of a simple C 3 -symmetric molecular system (TGP) with an intrinsic positive charge through a single-step Schiff base condensation. In a water−dimethyl sulfoxide (DMSO) solvent mixture (80:20 v/v), TGP molecules self-aggregate to form spherical nanoparticles with a positively charged surface that displays efficient fluorescence owing to the aggregation-induced emission (AIE) phenomenon. Both Gram-positive and Gram-negative bacteria could be effectively detected through "turn-off" fluorescence spectroscopy as the electrostatic interaction of the resultant nanoaggregates with the negatively charged bacterial surface induced quenching of fluorescence of the nanoparticles. The fluorescence analysis and steady-state lifetime studies of TGP nanoparticles suggest that a nonradiative decay through photoinduced electron transfer from the nanoparticles to the bacterial surface leads to effective fluorescence quenching. Further, the TGP nanoaggregates demonstrate potent antimicrobial activity against microbes such as multidrug-resistant bacteria and fungi at a concentration as low as 74 μg/mL. A combination of factors including ionic surface characteristics of the nanoparticles for strong electrostatic binding on the bacterial surface followed by possible photoinduced electron transfer from the nanoaggregates to the bacterial membrane and enhanced oxidative stress in the membrane resulting from reactive oxygen species (ROS) generation is found accountable for the high antimicrobial activity of the TGP nanoparticles. The effective disruption of membrane integrity in both Gram-positive and Gram-negative bacteria upon interaction with the nanoaggregates can be observed from field emission scanning electron microscopy (FESEM) studies. The development of simple pathways for the molecular design of multifunctional broad-spectrum antimicrobial systems for rapid and real-time detection, washfree imaging, and eradication of drug-resistant microbes might be crucial to combat pathogenic agents.
A series of environment-friendly cationic dye adsorbents, namely, pH-sensitive superparamagnetic hydrogel nanocomposite AA-VSA-P/SPIONs systems with different concentrations of superparamagnetic iron oxide nanoparticles (SPIONs; 1.2, 3.2, and 5.2 wt %), was synthesized by free-radical polymerization reaction using two pH-sensitive monomers, acrylic acid (AA) and vinylsulfonic acid (VSA), in an optimum ratio, in the presence of presynthesized SPIONs. The structural properties, thermal stability, and chemical configuration of AA-VSA-P/SPIONs systems with different weight percentages of SPIONs were characterized by XRD, TGA, Raman spectroscopy, and FTIR spectroscopy. The systems show substantial efficiency as dye adsorbents for removing cationic dyes (MB dye) from aqueous solution in neutral to alkaline medium. Further, these systems exhibit easy magnetic separation capabilities from aqueous solutions after dye adsorption, even for a very low weight percentage of SPIONs. The adsorption kinetics, mechanism, and isotherms of these systems were evaluated. The study suggests consistency with the pseudo-second-order kinetic model, following an intraparticle diffusion mechanism, where the heterogeneous surface of the system having different activation energies for adsorption plays the crucial role in dye adsorption via chemisorption for higher pH medium, which was further substantiated by excellent data fit with the Freundlich isotherm model. Biocompatibility and regeneration-ability studies establish the environment-friendliness and cost effectivity of the system.
M24B peptidases cleaving Xaa‐Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases‐P. Bacteria have small aminopeptidases‐P (36‐39 kDa), which are diverged from canonical aminopeptidase‐P of Escherichia coli (50 kDa). Structure‐function studies of small aminopeptidases‐P are lacking. We report crystal structures of small aminopeptidases‐P from E. coli and Deinococcus radiodurans, and report substrate‐specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases‐P, structurally close to small prolidases except for absence of dipeptide‐selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771‐2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase‐P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases‐P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases‐P from other M24B peptidases.
Xaa-Pro dipeptidase (XPD; prolidase; EC 3.4.13.9) specifically hydrolyzes dipeptides with a prolyl residue at the carboxy-terminus. Xanthomonas spp. possess two different isoforms of XPD (48 and 43 kDa) which share $24% sequence identity. The XPD of 43 kDa in size (XPD43) from Xanthomonas spp. is unusual as it lacks the strictly conserved tyrosine residue (equivalent to Tyr387 in Escherichia coli aminopeptidase P) that is suggested to be important in the proton-shuttle transfer required for catalysis in the M24B (MEROPS) family. Here, the crystallization and preliminary X-ray analysis of XPD43 from X. campestris (GenBank accession No. NP_637763) are reported. Recombinant XPD43 was crystallized using the microbatch-under-oil technique. Diffraction data were collected on the recently commissioned protein crystallography beamline (PX-BL21) at the Indian synchrotron (Indus-2, 2.5 GeV) to 1.83 Å resolution with 100% completeness. The crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 84.32, b = 105.51, c = 111.35 Å . Two monomers are expected to be present in the asymmetric unit of the crystal, corresponding to a solvent content of 58%. Structural analysis of XPD43 will provide new insights into the role of the conserved residues in catalysis in the M24B family.
Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of an N-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the -amyloid peptides implicated in Alzheimer's disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium, Deinococcus radiodurans (APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space group P2 1 , with unit-cell parameters a = 77.6, b = 189.6, c = 120.4 Å , = 108.4. A Matthews coefficient of 2.89 Å 3 Da À1 corresponds to four monomers, each with a molecular mass of $73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme in D. radiodurans.
Mannose-binding lectin-associated serine protease-1 (MASP-1) is known to interact with complement and coagulation pathways. Recently it was reported that MASP-1 interacts with the fibrinolytic system but details remain unclear. The objective of the study is to find MASP-1 substrates that participate in the fibrinolytic system. Commercially available fibrinogen might contain some impurities. Fibrinogen was treated with MASP-1 followed by analysis on SDS–PAGE and the obtained cleaved fragments were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight. Functional analysis of identified substrate was confirmed by fluorogenic and turbidimetric assay. Statistical analysis was done by using the Student t test. This study reports that plasminogen and plasma fibronectin are two hitherto unknown substrates of MASP-1. Conversion of plasminogen to plasmin like molecule by MASP-1 was confirmed by cleavage of plasmin specific substrate and digestion of fibrin clot. The role of MASP-1 in clot dissolution was confirmed by turbidity assay. Our study shows that MASP-1 selects plasminogen over fibrinogen to be a preferable substrate. MASP-1 promotes the fibrinolytic activity by the generation of plasmin like molecule from plasminogen and further destabilizes the clot by digestion of plasma fibronectin.
Xaa-Pro peptidases (XPP) are dinuclear peptidases of MEROPS M24B family that hydrolyze Xaa-Pro iminopeptide bond with a trans-proline at the second position of the peptide substrate. XPPs specific towards dipeptides are called prolidases while those that prefer longer oligopeptides are called aminopeptidases P. Though XPPs are strictly conserved in bacterial and archaeal species, the structural and sequence features that distinguish between prolidases and aminopeptidases P are not always clear. Here, we report 1.4 Å resolution crystal structure of a novel XPP from Deinococcus radiodurans (XPPdr). XPPdr forms a novel dimeric structure via unique dimer stabilization loops of N-terminal domains such that their C-terminal domains are placed far apart from each other. This novel dimerization is also the consequence of a different orientation of N-terminal domain in XPPdr monomer than those in other known prolidases. The enzymatic assays show that it is a prolidase with broad substrate specificity. Our structural, mutational, and molecular dynamics simulation analyses show that the conserved Arg46 of N-terminal domain is important for the dipeptide selectivity. Our BLAST search found XPPdr orthologs with conserved sequence motifs which correspond to unique structural features of XPPdr, thus identify a new subfamily of bacterial prolidases.
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