Structural variation in bacterial glyoxalase I enzymes: investigation of the metalloenzyme glyoxalase I from Clostridium acetobutylicum

The glyoxalase system catalyzes the conversion of toxic, metabolically produced α-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn(2+) and non-Zn(2+) activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Gram-positive bacteria that has been fully characterized as to its three-dimensional structure and its detailed metal specificity. It is a Ni(2+)/Co(2+)-activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn(2+)-bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements.     

High-level expression and one-step purification of recombinant dengue virus type 2 envelope domain III protein in Escherichia coli

Dengue virus infection poses a serious global public health threat for which there is currently no therapy or a licensed vaccine. The domain III of the dengue virus encoded envelope protein, which carries multiple conformation-dependent neutralizing epitopes, is critical for virus infectivity. We have expressed and purified recombinant domain III of dengue virus type-2 envelope, without the aid of a carrier protein in Escherichia coli. A 6x His tag was inserted at the N terminus to facilitate its one-step purification. The protein was overexpressed in the form of insoluble inclusion bodies, which were solubilized under highly denaturing conditions and then subjected to a previously optimized arginine-mediated renaturation protocol. We purified recombinant domain III protein to near homogeneity by Ni-NTA affinity chromatography and obtained yields of approximately 30 mg/L. The purified protein was recognized in Western analyses by monoclonal antibodies specific for the 6x His tag as well as the 3H5 neutralizing epitope known to reside in domain III. The authenticity of the recombinant protein was also verified in a sandwich ELISA designed to specifically and simultaneously identify the 6x His tag and the 3H5 epitope. In addition, murine and human polyclonal sera also recognized the recombinant protein. The in vitro refolded recombinant protein preparation was biologically functional. It could effectively protect cells in culture against dengue virus type-2 infection, apparently by blocking the virus from binding to host cells. This expression/purification strategy has the potential for inexpensive scale-up and may prove to be useful for dengue diagnostics and vaccine development efforts.     

SAR of 3,4-dihydropyrido[3,2-d]pyrimidone p38 inhibitors

Development for a class of potent 3,4-dihydropyrido(3,2-d)pyrimidone inhibitors of p38a MAP kinase is described. Modification of N-1 aryl and C-6 arylsulfide in 3,4-dihydropyrido(3,2-d)pyrimidone analogues for the interaction with the hydrophobic pockets in p38 active site is also discussed.     

A novel mechanism for Clostridium botulinum neurotoxin inhibition

Clostridium botulinum neurotoxins are zinc endopeptidase proteins responsible for cleaving specific peptide bonds of proteins of neuroexocytosis apparatus. The ability of drugs to interfere with toxin's catalytic activity is being evaluated with zinc chelators and metalloprotease inhibitors. It is important to develop effective pharmacological treatment for the intact holotoxin before the catalytic domain separates and enters the cytosol. We present here evidence for a novel mechanism of an inhibitor binding to the holotoxin and for the chelation of zinc from our structural studies on Clostridium botulinum neurotoxin type B in complex with a potential metalloprotease inhibitor, bis(5-amidino-2-benzimidazolyl)methane, and provide snapshots of the reaction as it progresses. The binding and inhibition mechanism of this inhibitor to the neurotoxin seems to be unique for intact botulinum neurotoxins. The environment of the active site rearranges in the presence of the inhibitor, and the zinc ion is gradually removed from the active site and transported to a different site in the protein, probably causing loss of catalytic activity.     

Fine Mapping and Functional Analysis of the Multiple Sclerosis Risk Gene CD6

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2^nd SRCR domain with susceptibility to MS (P _{max(T) permutation} = 1×10⁻⁴). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. – CD4⁺ naïve cells, P = 0.0001; CD8⁺ naïve cells, P<0.0001; CD4⁺ and CD8⁺ central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4⁺ and CD8⁺ T cells.     

Performance of inverse anaerobic fluidized bed reactor for treating high strength organic wastewater during start-up phase

The aim of this work is to report on the physical characteristics of carrier material (perlite), biomass growth on the carrier material and the biogas production during an apparent steady state period in an inverse anaerobic fluidized bed reactor (IAFBR) for treating high strength organic wastewater. Before starting up the reactor, physical properties of the carrier material were determined. One millimeter diameter perlite particle is found to have a wet specific density of 295 kg/m(3) with specific surface area of 7.010 m(2)/g. This material has provided a good surface for biomass attachment and development. The biofilm concentration (in terms of attached volatile solids (AVS)) attached to carrier material was found to be 0.66 g(AVS)/g(solid). Most particles have been covered with a thin biofilm of uniform thickness. Once the inverse anaerobic fluidized bed system reached the steady state, the organic load was increased step wise by reducing hydraulic retention time (HRT) from 2 days to 0.16 day, while maintaining the constant feed of chemical oxygen demand (COD) concentration. This system has achieved 84% COD removal and reached the biogas production of 13.22 l/l/d at an organic loading rate (OLR) of 35 kgCOD/m(3)/d.