Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B peptidases.

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.

Crystal structure of a novel prolidase from Deinococcus radiodurans identifies new subfamily of bacterial prolidases.

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.

The members of M20D peptidase subfamily from Burkholderia cepacia, Deinococcus radiodurans and Staphylococcus aureus (HmrA) are carboxydipeptidases, primarily specific for Met-X dipeptides.

Three members of peptidase family M20D from Burkholderia cepacia (BcepM20D; Uniprot accession no. A0A0F7GQ23), Deinococcus radiodurans R1 (DradM20D; Uniprot accession no. Q9RTP6) and Staphylococcus aureus (HmrA; Uniprot accession no. Q99Q45) were characterized in terms of their preference for various substrates. The results thus reveal that all the enzymes including HmrA lack endopeptidase as well as aminopeptidase activities and possess strong carboxypeptidase activity. Further, the amidohydrolase activity exerted on other substrates like N-Acetyl-Amino acids, N-Carbobenzoxyl-Amino acids and Indole acetic acid (IAA)-Amino acids is due to the ability of these enzymes to accommodate different types of chemical groups other than the amino acid at the S1 pocket. Further, data on peptide hydrolysis strongly suggests that all the three enzymes are primarily carboxydipeptidases exhibiting highest catalytic efficiency (kcat/Km 5-36 × 10(5) M(-1) s(-1)) for Met-X substrates, where -X could be Ala/Gly/Ser/Tyr/Phe/Leu depending on the source organism. The supportive evidence for the substrate specificities was also provided with the molecular docking studies carried out using structure of SACOL0085 and homology modelled structure of BcepM20D. The preference for different substrates, their binding at active site of the enzyme and possible role of these enzymes in recycling of methionine are discussed in this study.

Predicting immune response targets in orthoflaviviruses through sequence homology and computational analysis.

CONTEXT: Flaviviruses cause severe encephalitic or hemorrhagic diseases in humans. Its members, Kyasanur forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (ALKV), cause hemorrhagic fever and are prevalent in India and Saudi Arabia, respectively, while the tick-borne encephalitis virus (TBEV) causes a dangerous encephalitic infection in Europe and Asia. However, little information is available about the targets of immune responses for these deadly viruses. Here, we predict potential antigenic peptide epitopes of viral envelope protein for inducing a cell-mediated and humoral immune response. METHODS: Using the Immune Epitope Database and Analysis Resource (IEDB-AR), we identified 13 MHC-I and two MHC-II dominant conserved epitopes in KFDV and ALKV and six MHC-I and three MHC-II epitopes in TBEV envelope proteins. Parallelly, we also predicted B-cell linear and discontinuous envelope protein epitopes for these viruses. Interestingly, the epitopes are conserved in all three viral envelope proteins. Further, the discontinuous epitopes are structurally compared with the available DENV, ZIKV, WNV, TBEV, and LIV envelope protein antibody structures. Overall structural comparison analyses highlight (i) lateral ridge epitope in the ED-III domain of E protein, and (ii) envelope dimer epitope (EDE) could be targeted for developing potent vaccine candidates as well as therapeutic antibody production. Moreover, existing structural and biochemical functions of the same epitopes in homologous viruses are predicted to have a reduced antibody-dependent enhancement (ADE) effect on flaviviral infection.

Mannose-binding lectin-associated serine protease-1 cleaves plasminogen and plasma fibronectin: prefers plasminogen over known fibrinogen substrate.

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.

Cationic Organic Nanoaggregates as AIE Luminogens for Wash-Free Imaging of Bacteria and Broad-Spectrum Antimicrobial Application.

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 C3-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, wash-free imaging, and eradication of drug-resistant microbes might be crucial to combat pathogenic agents.