ABSTRACT
Ba0331 is a putative polysaccharide deacetylase from Bacillus anthracis, the etiological agent of the disease anthrax, that contributes to adaptation of the bacterium under extreme conditions and to maintenance of the cell shape. In the present study, the crystal structure of Ba0331 was determined at 2.6â Å resolution. The structure consists of two domains: a fibronectin type 3-like (Fn3-like) domain and a NodB catalytic domain. The latter is present in all carbohydrate esterase family 4 enzymes, while a comparative analysis of the Fn3-like domain revealed structural plasticity despite the retention of the conserved Fn3-like domain characteristics.
Subject(s)
Amidohydrolases/chemistry , Amidohydrolases/genetics , Bacillus anthracis/enzymology , Gene Expression , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Models, Molecular , Protein Domains , Static Electricity , Zinc/metabolismABSTRACT
Peptidoglycan N-acetylglucosamine (GlcNAc) deacetylases (PGNGdacs) from bacterial pathogens are validated targets for the development of novel antimicrobial agents. In this study we examined the in vitro inhibition of hydroxamate ligand N-hydroxy-4-(naphthalene-1-yl)benzamide (NHNB), a selective inhibitor of histone deacetylases-8 (HDAC8), against two PGNGdacs namely BC1974 and BC1960 from B. cereus, highly homologous to BA1977 and BA1961 of B. anthracis, respectively. Kinetic analysis showed that this compound functions as a competitive inhibitor of both enzymes with apparent Ki's of 8.7⯵M (for BC1974) and 66⯵M (for BC1960), providing thus the most potent CE4 inhibitor reported to date. NHNB was tested in antibacterial assays and showed bactericidal activity against both examined pathogens acting as a multi-target drug. This compound can serve as lead for the development of inhibitors targeting the conserved active sites of the multiple polysaccharide deacetylases (PDAs) of both pathogens.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Bacillus anthracis/drug effects , Bacillus cereus/drug effects , Bacterial Proteins/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/chemistry , Amidohydrolases/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/chemical synthesis , Bacillus anthracis/enzymology , Bacillus cereus/enzymology , Bacterial Proteins/metabolism , Binding Sites , Catalytic Domain , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Microbial Sensitivity Tests , Molecular Dynamics Simulation , Sequence AlignmentABSTRACT
Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) ß-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.
Subject(s)
Amidohydrolases/metabolism , Anthrax/microbiology , Bacillus anthracis/cytology , Bacillus anthracis/enzymology , Bacterial Proteins/metabolism , Cell Wall/metabolism , Osmosis/physiology , Stress, Physiological , Amidohydrolases/chemistry , Amidohydrolases/genetics , Amino Acid Sequence , Anthrax/genetics , Anthrax/metabolism , Bacillus anthracis/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Blotting, Western , Cloning, Molecular , Crystallography, X-Ray , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Molecular Sequence Data , Peptidoglycan/metabolism , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Salt Tolerance , Sequence Homology, Amino AcidABSTRACT
Peptidoglycan deacetylases (PGNG-dacs) belong to the Carbohydrate Esterase Family 4 (CE4) and have been described as required for bacterial evasion to lysozyme and innate immune responses. Interestingly, there is an unusual occurrence of 10 putative polysaccharide deacetylases, including five PGNG-dacs, in the Bacillus sp. genomes, especially B. cereus and B. anthracis. To elucidate the physiological role of these multiple deacetylases, we employed genetic analysis and protein localization studies of five putative PGNG-dacs from B. anthracis as well as biochemical analysis of their corresponding homologues from B. cereus. Our data confirm that three enzymes are PGNG-dacs. While BA1977, associated with lateral peptidoglycan synthesis, is a bona fide peptidoglycan deacetylase involved in resistance to host lysozyme and required for full virulence, BA1961 and BA3679 participate in the biogenesis of the peptidoglycan during both elongation and cell division. Furthermore, two enzymes are important for neutral polysaccharide attachment to PG and consequently anchoring of S-layer proteins (BA5436) and for polysaccharide modification (BA2944). Our results provide novel and fundamental insights into the function of polysaccharide deacetylases in a major bioterrorism agent.