The Bacillus subtilis signaling protein SpoIVB defines a new family of serine peptidases

The protein SpoIVB plays a key role in signaling in the sigma (K) checkpoint of Bacillus subtilis. This regulatory mechanism coordinates late gene expression during development in this organism and we have recently shown SpoIVB to be a serine peptidase. SpoIVB signals by transiting a membrane, undergoing self-cleavage, and then by an unknown mechanism activating a zinc metalloprotease, SpoIVFB, which cleaves pro-sigma (K) to its active form, sigma (K), in the outer mother cell chamber of the developing cell. In this work we have characterized the serine peptidase domain of SpoIVB. Alignment of SpoIVB with homologues from other spore formers has allowed site-specific mutagenesis of all potential active site residues within the peptidase domain. We have defined the putative catalytic domain of the SpoIVB serine peptidase as a 160-amino-acid residue segment at the carboxyl terminus of the protein. His236 and Ser378 are the most important residues for proteolysis, with Asp363 being the most probable third member of the catalytic triad. In addition, we have shown that mutations at residues Asn290 and His394 lead to delayed signaling in the sigma (K) checkpoint. The active site residues suggest that SpoIVB and its homologues from other spore formers are members of a new family of serine peptidases of the trypsin superfamily

Bacillus subtilis regulatory protein GerE

GerE is the latest-acting of a series of factors which regulate gene expression in the mother cell during sporulation in Bacillus. The gene encoding GerE has been cloned from B. subtilis and overexpressed in Escherichia coli. Purified GerE has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. The small plate-like crystals belong to the monoclinic space group C2 and diffract beyond 2.2 Angstrom resolution with a synchrotron radiation X-ray source

Structure of d(TGCGCA)(2) and a comparison with other DNA Hexamers

The X-ray crystal structure of d(TGCGCA)(2) has been determined at 120 K to a resolution of 1.3 Angstrom. Hexamer duplexes, in the Z-DNA conformation, pack in an arrangement similar to the 'pure spermine form' [Egli et al. (1991). Biochemistry, 30, 11388-11402] but with significantly different cell dimensions. The phosphate backbone exists in two equally populated discrete conformations at one nucleotide step, around phosphate 11. The structure contains two ordered cobalt hexammine molecules which have roles in stabilization of both the Z-DNA conformation of the duplex and in crystal packing. A comparison of d(TGCGCA)(2) with other Z-DNA hexamer structures available in the Nucleic Acid Database illustrates the elusive nature of crystal packing. A review of the interactions with the metal cations Na+, Mg2+ and Co3+ reveals a relatively small proportion of phosphate binding and that close contacts between metal ions are common. A prediction of the water structure is compared with the observed pattern in the reported structure

Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium

Crystallization of YIoQ, a GTPase of unknown function essential for Bacillus subtilis viability

YLoQ is a putative ATP/GTP-binding protein of unknown function identified from the complete sequence of the Bacillus subtilis genome. A gene-knockout programme established that yloQ is one of a set of some 270 indispensable genes for the viability of this organism. Crystals of YloQ have been grown from HEPES-buffered solutions at pH 7.5 containing polyethylene glycol and diffraction data have been collected extending to 2.5 Angstrom spacing

Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding

Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k(cat)/K-m values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K-s. Values of k(cat/)K(m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k(cat)/K-m for the CySB71 mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 X resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S-1 subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity

Crystallization of the oligopeptide-binding protein AppA from Bacillus subtilis

AppA is the membrane-anchored extracellular receptor component of an ABC transporter responsible for the uptake of oligopeptides into Bacillus subtilis. AppA has been overexpressed as a cleavable maltose-binding protein fusion in Escherichia coli. Following removal of the fusion portion, AppA has been crystallized from morpholino-ethanesulfonic acid-buffered solutions at pH 6.5 containing polyethylene glycol and zinc acetate. A complete X-ray diffraction data set extending to 2.3 Angstrom spacing has been collected

Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

The crystallization of three catalytically inactive mutants of penicillin Vacylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide threedimensional structures of precursor PVA forms, plus open a route to the study of enzyme-substrate complexes for this industrially important enzyme

Soil Mixing with Steam and Zero Valent Iron Remediation Performance Monitoring Results at Wilson Corner, Kennedy Space Center, Florida

The National Aeronautics and Space Administration (NASA), Kennedy Space Center (KSC), Remediation Program implemented a groundwater interim measure between September 2014 and February 2015, to remediate a trichloroethene (TCE) groundwater source area at the Wilson Corners site. The groundwater plume is associated with the release of chlorinated solvents (specifically TCE) from historic precision cleaning and laboratory operations. A summary of the groundwater interim measure using soil mixing with steam and zero valent iron (also referred to as large diameter auger [LDA]) was presented at the 2015 Florida Remediation Conference. The objective of this presentation is to provide performance monitoring results

The crystal structure of superoxide dismutase from Plasmodium falciparum

Background: Superoxide dismutases (SODs) are important enzymes in defence against oxidative stress. In Plasmodium falciparum, they may be expected to have special significance since part of the parasite life cycle is spent in red blood cells where the formation of reactive oxygen species is likely to be promoted by the products of haemoglobin breakdown. Thus, inhibitors of P. falciparum SODs have potential as anti-malarial compounds. As a step towards their development we have determined the crystal structure of the parasite's cytosolic iron superoxide dismutase. Results: The cytosolic iron superoxide dismutase from P. falciparum (PfFeSOD) has been overexpressed in E. coli in a catalytically active form. Its crystal structure has been solved by molecular replacement and refined against data extending to 2.5 angstrom resolution. The structure reveals a two-domain organisation and an iron centre in which the metal is coordinated by three histidines, an aspartate and a solvent molecule. Consistent with ultracentrifugation analysis the enzyme is a dimer in which a hydrogen bonding lattice links the two active centres. Conclusion: The tertiary structure of PfFeSOD is very similar to those of a number of other iron-and manganese-dependent superoxide dismutases, moreover the active site residues are conserved suggesting a common mechanism of action. Comparison of the dimer interfaces of PfFeSOD with the human manganese-dependent superoxide dismutase reveals a number of differences, which may underpin the design of parasite-selective superoxide dismutase inhibitors