ABSTRACT
Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified ßαßßαßα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2.
Subject(s)
Burkholderia pseudomallei , Burkholderia pseudomallei/genetics , Arginine , Aspartic Acid , Catalysis , EndonucleasesABSTRACT
TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05â Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63â Å.
Subject(s)
Bacterial Proteins/chemistry , Receptors, GABA-A/chemistry , Streptomyces/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Ethylene Glycol/chemistry , Gene Expression , Glycerol/chemistry , Plasmids/chemistry , Plasmids/metabolism , Protein Conformation, alpha-Helical , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Streptomyces/metabolism , X-Ray DiffractionABSTRACT
Melioidosis is an infectious disease caused by the pathogenic bacterium Burkholderia pseudomallei. Whole-genome sequencing revealed that the B. pseudomallei genome includes 5855 coding DNA sequences (CDSs), of which â¼25% encode hypothetical proteins. A pathogen-associated hypothetical protein, BPSL1038, was overexpressed in Escherichia coli, purified and crystallized using vapour-diffusion methods. A BPSL1038 protein crystal that grew using sodium formate as precipitant diffracted to 1.55â Å resolution. It belonged to space group C2221, with unit-cell parameters a = 85.36, b = 115.63, c = 46.73â Å. The calculated Matthews coefficient (VM) suggests that there are two molecules per asymmetric unit, with a solvent content of 48.8%.
