ABSTRACT
Nipah Virus (NiV) has been designated as a priority disease with an urgent need for therapeutic development by World Health Organization. The monoclonal antibody m102.4 binds to the immunodominant NiV receptor-binding glycoprotein (GP), and potently neutralizes NiV, indicating its potential as a therapeutic agent. Although the co-crystal structure of m102.3, an m102.4 derivative, in complex with the GP of the related Hendra Virus (HeV) has been solved, the structural interaction between m102.4 and NiV is uncharacterized. Herein, we used structure-guided alanine-scanning mutagenesis to map the functional epitope and paratope residues that govern the antigen-antibody interaction. Our results revealed that the binding of m102.4 is mediated predominantly by two residues in the HCDR3 region, which is unusually small for an antibody-antigen interaction. We performed computational docking to generate a structural model of m102.4-NiV interaction. Our model indicates that m102.4 targets the common hydrophobic central cavity and a hydrophilic rim on the GP, as observed for the m102.3-HeV co-crystal, albeit with Fv orientation differences. In summary, our study provides insight into the m102.4-NiV interaction, demonstrating that structure-guided alanine-scanning and computational modeling can serve as the starting point for additional antibody reengineering (e.g. affinity maturation) to generate potential therapeutic candidates.
Subject(s)
Alanine/genetics , Antibodies, Monoclonal/metabolism , Computer Simulation , Glycoproteins/metabolism , Henipavirus Infections/virology , Nipah Virus/metabolism , Viral Envelope Proteins/metabolism , Alanine/chemistry , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/immunology , Antigen-Antibody Complex/metabolism , Epitopes/immunology , Glycoproteins/chemistry , Glycoproteins/genetics , Henipavirus Infections/immunology , Henipavirus Infections/metabolism , Humans , Mutagenesis, Site-Directed , Nipah Virus/immunology , Nipah Virus/isolation & purification , Protein Structural Elements/immunology , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/geneticsABSTRACT
The esterase B (EstB) from Sphingobium sp. SM42, which was previously reported to be active towards dibutyl phthalate, can cleave some small aromatic ring side chains from cephalosporin derivatives. A new name, de-arenethiolase, has been proposed to represent this activity. We present the in vitro characterization of the activity of purified EstB toward cephalosporin substrates. Interestingly, EstB was highly active against cefoperazone and cefazolin resulting in 83 and 67% decreases in killing zone diameter, respectively. EstB also demonstrated a moderate activity towards ceftriaxone (18%) and cefotaxime (16%) while exhibiting no activity against cephalosporin C and cefixime. HPLC analysis indicated that EstB catalyzed the cleavage of the C-S bond found in cephalosporin derivatives to release the corresponding free aromatic ring side chains.
Subject(s)
Cephalosporins/metabolism , Serine Endopeptidases/metabolism , Sphingobacterium/enzymology , Bacterial Proteins/metabolism , Bacteroidetes/enzymology , Cefotaxime/metabolism , Ceftriaxone/metabolism , Cephalosporins/antagonists & inhibitors , Substrate SpecificitySubject(s)
Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Butyrates/isolation & purification , Butyrates/pharmacology , Streptomyces/chemistry , Anti-Infective Agents/chemistry , Anti-Infective Agents/isolation & purification , Anti-Infective Agents/pharmacology , Antineoplastic Agents/chemistry , Butyrates/chemistry , Candida albicans/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Gram-Positive Bacteria/drug effects , Humans , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Microbial Sensitivity Tests , Models, Molecular , Molecular StructureABSTRACT
Proteus sp. SW1 was found to produce an extracellular solvent tolerant lipase. The gene, lipA, encoding a bacterial lipase, was cloned from total Proteus sp. SW1 DNA. lipA was predicted to encode a 287 amino acid protein of 31.2 kDa belonging to the Group I proteobacterial lipases. Purified His-tagged LipA exhibited optimal activity at pH 10.0 and 55°C. It was highly stable in organic solvents retaining 112% of its activity in 100% isopropanol after 24 h, and exhibited more than 200% of its initial activity upon exposure to 60% acetone, ethanol, and hexane for 18 h. Biodiesel synthesis reactions, using a single step addition of 13% an acyl acceptor ethanol, showed that LipA was highly effective at converting palm oil into biodiesel.
