Characterization of a pseudomonad 2-nitrobenzoate nitroreductase and its catabolic pathway-associated 2-hydroxylaminobenzoate mutase and a chemoreceptor involved in 2-nitrobenzoate chemotaxis

Pseudomonas fluorescens strain KU-7 is a prototype microorganism that metabolizes 2-nitrobenzoate (2-NBA) via the formation of 3-hydroxyanthranilate (3-HAA), a known antioxidant and reductant. The initial two steps leading to the sequential formation of 2-hydroxy/aminobenzoate and 3-HAA are catalyzed by a NADPH-dependent 2-NBA nitroreductase (NbaA) and 2-hydroxylaminobenzoate mutase (NbaB), respectively. The 216-amino-acid protein NbaA is 78% identical to a plasmid-encoded hypothetical conserved protein of Polaromonas strain JS666; structurally, it belongs to the homodimeric NADH:flavin mononucleotide (FMN) oxidoreductase-like fold family. Structural modeling of complexes with the flavin, coenzyme, and substrate suggested specific residues contributing to the NbaA catalytic activity, assuming a ping-pong reaction mechanism. Mutational analysis supports the roles of Asn40, Asp76, and Glu113, which are predicted to form the binding site for a divalent metal ion implicated in FMN binding, and a role in NADPH binding for the 10-residue insertion in the beta5-alpha2 loop. The 181-amino-acid sequence of NbaB is 35% identical to the 4-hydroxylaminobenzoate lyases (PnbBs) of various 4-nitrobenzoate-assimilating bacteria, e.g., Pseudomonas putida strain TW3. Coexpression of nbaB with nbaA in Escherichia coli produced a small amount of 3-HAA from 2-NBA, supporting the functionality of the nbaB gene. We also showed by gene knockout and chemotaxis assays that nbaY, a chemoreceptor NahY homolog located downstream of the nbaA gene, is responsible for strain KU-7 being attracted to 2-NBA. NbaY is the first chemoreceptor in nitroaromatic metabolism to be identified, and this study completes the gene elucidation of 2-NBA metabolism that is localized within a 24-kb chromosomal locus of strain KU-7.     

Molecular dynamics-solvated interaction energy studies of protein-protein interactions: the MP1-p14 scaffolding complex

Using the MP1-p14 scaffolding complex from the mitogen-activated protein kinase signaling pathway as model system, we explored a structure-based computational protocol to probe and characterize binding affinity hot spots at protein-protein interfaces. Hot spots are located by virtual alanine-scanning consensus predictions over three different energy functions and two different single-structure representations of the complex. Refined binding affinity predictions for select hot-spot mutations are carried out by applying first-principle methods such as the molecular mechanics generalized Born surface area (MM-GBSA) and solvated interaction energy (SIE) to the molecular dynamics (MD) trajectories for mutated and wild-type complexes. Here, predicted hot-spot residues were actually mutated to alanine, and crystal structures of the mutated complexes were determined. Two mutated MP1-p14 complexes were investigated, the p14(Y56A)-mutated complex and the MP1(L63A,L65A)-mutated complex. Alternative ways to generate MD ensembles for mutant complexes, not relying on crystal structures for mutated complexes, were also investigated. The SIE function, fitted on protein-ligand binding affinities, gave absolute binding affinity predictions in excellent agreement with experiment and outperformed standard MM-GBSA predictions when tested on the MD ensembles of Ras-Raf and Ras-RalGDS protein-protein complexes. For wild-type and mutant MP1-p14 complexes, SIE predictions of relative binding affinities were supported by a yeast two-hybrid assay that provided semiquantitative relative interaction strengths. Results on the MP1-mutated complex suggested that SIE predictions deteriorate if mutant MD ensembles are approximated by just mutating the wild-type MD trajectory. The SIE data on the p14-mutated complex indicated feasibility for generating mutant MD ensembles from mutated wild-type crystal structure, despite local structural differences observed upon mutation. For energetic considerations, this would circumvent costly needs to produce and crystallize mutated complexes. The sensitized protein-protein interface afforded by the p14(Y56A) mutation identified here has practical applications in screening-based discovery of first-generation small-molecule hits for further development into specific modulators of the mitogen-activated protein kinase signaling pathway.     

Spatial heterogeneity in sediment-associated bacterial and eukaryotic communities in a landfill leachate-contaminated aquifer

Heterogeneity in eukaryotic and bacteria community structure in surface and subsurface sediment samples downgradient of the Banisveld landfill (The Netherlands) was studied using a culturing-independent molecular approach. Along a transect covering the part of the aquifer most polluted by landfill leachate, sediment was sampled at 1-m depth intervals, until a depth of 5.5 m, at four distances from the landfill. Two drillings were placed in a nearby clean area as a reference. Denaturing gradient gel electrophoresis banding patterns revealed high bacterial and eukaryotic diversity and complex community structures. Bacteria and eukaryotic community profiles in polluted samples grouped different from those in clean samples. Bacteria community profiles in surface samples clustered together and separately from subsurface community profiles. Subsurface bacteria profiles clustered in a location-specific manner. Eukaryotic community structure did not significantly relate to distance from the landfill or depth. No significant spatial autocorrelation of bacteria or eukaryotic communities was observed over 1-m depth intervals per sampling location. Spatial heterogeneity in sediment-associated bacterial communities appears to be much larger than in groundwater. We discuss how on the one hand, spatial heterogeneity may complicate the assessment of microbial community structure and functioning, while on the other it may provide better opportunities for natural attenuation.     

Binding site-based classification of coronaviral papain-like proteases

The coronavirus replicase gene encodes one or two papain-like proteases (termed PL1pro and PL2pro) implicated in the N-terminal processing of the replicase polyprotein and thus contributing to the formation of the viral replicase complex that mediates genome replication. Using consensus fold recognition with the 3D-JURY meta-predictor followed by model building and refinement, we developed a structural model for the single PLpro present in the severe acute respiratory syndrome coronavirus (SCoV) genome, based on significant structural relationships to the catalytic core domain of HAUSP, a ubiquitin-specific protease (USP). By combining the SCoV PLpro model with comparative sequence analyses we show that all currently known coronaviral PLpros can be classified into two groups according to their binding site architectures. One group includes all PL2pros and some of the PL1pros, which are characterized by a restricted USP-like binding site. This group is designated the R-group. The remaining PL1pros from some of the coronaviruses form the other group, featuring a more open papain-like binding site, and is referred to as the O-group. This two-group, binding site-based classification is consistent with experimental data accumulated to date for the specificity of PLpro-mediated polyprotein processing and PLpro inhibition. It also provides an independent evaluation of the similarity-based annotation of PLpro-mediated cleavage sites, as well as a basis for comparison with previous groupings based on phylogenetic analyses.     

Crystal structure of Methanobacterium thermoautotrophicum phosphoribosyl-AMP cyclohydrolase HisI

The metabolic pathway for histidine biosynthesis is interesting from an evolutionary perspective because of the diversity of gene organizations and protein structures involved. Hydrolysis of phosphoribosyl-AMP, the third step in the histidine biosynthetic pathway, is carried out by PR-AMP cyclohydrolase, the product of the hisI gene. The three-dimensional structure of PR-AMP cyclohydrolase from Methanobacterium thermoautotrophicum was solved and refined to 1.7 A resolution. The enzyme is a homodimer. The position of the Zn(2+)-binding site that is essential for catalysis was inferred from the positions of bound Cd(2+) ions, which were part of the crystallization medium. These metal binding sites include three cysteine ligands, two from one monomer and the third from the second monomer. The enzyme remains active when Cd(2+) is substituted for Zn(2+). The likely binding site for Mg(2+), also necessary for activity in a homologous cyclohydrolase, was also inferred from Cd(2+) positions and is comprised of aspartic acid side chains. The putative substrate-binding cleft is formed at the interface between the two monomers of the dimer. This fact, combined with the localization of the Zn(2+)-binding site, indicates that the enzyme is an obligate dimer.     

The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.     

Protein microarray for complex apoptosis monitoring of dysplastic oral keratinocytes in experimental photodynamic therapy

Background

Photodynamic therapy is an alternative treatment of muco-cutaneous tumors that uses a light source able to photoactivate a chemical compound that acts as a photosensitizer. The phthalocyanines append to a wide chemical class that encompasses a large range of compounds; out of them aluminium-substituted disulphonated phthalocyanine possesses a good photosensitizing potential.

Results

The destructive effects of PDT with aluminium-substituted disulphonated phthalocyanine are achieved by induction of apoptosis in tumoral cells as assessed by flow cytometry analysis. Using protein microarray we evaluate the possible molecular pathways by which photodynamic therapy activates apoptosis in dysplastic oral keratinocytes cells, leading to the tumoral cells destruction. Among assessed analytes, Bcl-2, P70S6K kinase, Raf-1 and Bad proteins represent the apoptosis related biomolecules that showed expression variations with the greatest amplitude.

Conclusions

Up to date, the intimate molecular apoptotic mechanisms activated by photodynamic therapy with this type of phthalocyanine in dysplastic human oral keratinocytes are not completely elucidated. With protein microarray as high-throughput proteomic approach a better understanding of the manner in which photodynamic therapy leads to tumoral cell destruction can be obtained, by depicting apoptotic molecules that can be potentially triggered in future anti-tumoral therapies.     

Experimental calcification of HEMA-based hydrogels in the presence of albumin and a comparison to the in vivo calcification

The precipitation patterns and characteristics of calcium phosphate (CaP) phases deposited on HEMA-based hydrogels upon incubation in simulated body fluid (SBF-2) containing a protein (human serum albumin) have been investigated in relation to the calcification in an organic-free medium (SBF-1) and to that occurring after subcutaneous implantation in rats. In SBF-2, the deposits occurred exclusively as a peripheral layer on the surface of the hydrogels and consisted mainly of "precipitated hydroxyapatite", a species deficient in calcium and hydroxyl ions, similarly to the deposits formed on the implanted hydrogels, where the deposited layer was thicker. In SBF-1, the deposits were mainly of brushite type. There was no evidence that albumin penetrated the interstices of hydrogels. As the X-ray diffraction patterns of the CaP deposits generated in SBF-2 showed a similar nature with those formed on the implanted hydrogel, it was concluded that the calcification in SBF-2 can mimic to a reliable extent the calcification process taking place in a biological environment.     

Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate

Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a β-elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles.