An evolutionary non-conserved motif in Helicobacter pylori arginase mediates positioning of the loop containing the catalytic residue for catalysis.

The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (-153ESEEKAWQKLCSL165-) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122-Trp159-Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57-Glu155-Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy.

Functional characterization of the transcription regulator WhiB1 from Gordonia sp. IITR100.

WhiB is a transcription regulator which has been reported to be involved in the regulation of cell morphogenesis, cell division, antibiotic resistance, stress, etc., in several members of the family Actinomycetes. The present study describes functional characterization of a WhiB family protein, WhiB1 (protein ID: WP_065632651.1), from Gordonia sp. IITR100. We demonstrate that WhiB1 affects chromosome segregation and cell morphology in recombinant Escherichia coli, Gordonia sp. IITR100 as well as in Rhodococcus erythropolis. Multiple sequence alignment suggests that WhiB1 is a conserved protein among members of the family Actinomycetes. It has been reported that overexpression of WhiB1 leads to repression of the biodesulfurization operon in recombinant E. coli, Gordonia sp. IITR100 and R. erythropolis. A WhiB1-mut containing a point mutation Q116A in the DNA binding domain of WhiB1 led to partial alleviation of repression of the biodesulfurization operon. We show for the first time that the WhiB family protein WhiB1 is also involved in repression of the biodesulfurization operon by directly binding to the dsz promoter DNA.

Effect of the Macromolecular Crowding Agents on the Structure and Function of Human Arginase-I, a Therapeutically Important Enzyme.

Macromolecular crowding has been known to influence the structure and function of many enzymes through excluded volume effects and/or soft interactions. Here, we employed two synthetic macromolecular crowders, Dextrans and poly(ethylene glycol)s (PEGs) with varying molecular masses, to examine how they affected the structure and function of a therapeutically important enzyme, human arginase-I that catalyzes the conversion of l-arginine to l-ornithine and urea. Except at greater concentrations of Dextran 200, Dextrans were observed to slightly reduce the enzymatic activity, indicating that they exert their influence mainly through the excluded volume effects. Similar outcomes were seen with PEGs, with the exception of PEG 1000, where the activity decreased with increasing PEG concentrations, showing the maximum effect at a 20 g/L concentration. This finding suggests that the enzyme function is reduced by the soft interactions of this macromolecule with the enzyme, supported by the binding measurement. Secondary and local tertiary structures and thermodynamic stability were also affected, suggesting that PEG 1000 has an impact on the protein's structure. Furthermore, molecular dynamics simulation studies suggest that the catalytic pocket is disturbed, presumably by the unwinding of neighboring helix 9. As a result, the positioning of nearby Glu277 is altered, which prevents His141 and Glu277 from making contact. This hampers the proton transfer from the catalytic His141 to the intermediate species to form ornithine, a crucial step for the substrate hydrolysis reaction by this arginase. Overall, the knowledge gained from this study might be helpful for understanding how different enzymes work in a crowded/cellular environment.

Bending is required for activation of dsz operon by the TetR family protein (DszGR).

The dsz operon responsible for the biodesulfurization of organosulfurs is under the control of a 385 bp long promoter. Recently, a TetR family protein was identified which served as an activator of operon. Here we report that the TetR family protein (WP_058249973.1), named DszGR can specifically activate the dsz operon. Direct binding of the DszGR to DNA was observed at single molecule level by AFM. It was found that the binding of DszGR to the promoter DNA induces a bend by about âˆ¼40-50° degrees which may not be enough for the activation of the promoter. Thus, bendability in the promoter sequence was analyzed. The results show that the promoter has a curvature at around -235 and -200 bp with respect to dszA start codon. On mutating this region, a decrease in activity of the promoter was observed. Our results suggest that the DszGR protein binds to the upstream sequences and induces a bend, which is facilitated by further bending of the DNA which is required for dsz promoter activity. IHF binding site present in the promoter, and a significant reduction in desulphurization activity in the absence of either IHF subunits, suggested role of IHF in regulation of the dsz operon.

Characterization of DNA binding and ligand binding properties of the TetR family protein involved in regulation of dsz operon in Gordonia sp. IITR100.

Gordonia sp. IITR100 is a biodesulfurizing bacterium which can metabolize dibenzothiophene (DBT) to 2 hydroxybiphenyl in four steps via the 4S pathway. The genes involved in the metabolism are present in the form of an operon, dszABC, which gets activated by a TetR family protein. Here, we report the detailed characterization of the DNA binding and ligand binding property of the TetR family protein. The protein was found to be conserved across other desulfurizing organisms. The protein was purified and was found to exist as dimer. The presence of ligand binding site was identified by docking studies and the structural changes in the protein upon ligand binding were determined by CD spectroscopy and tryptophan fluorescence. Further, it was determined that this protein binds to an imperfect palindromic DNA sequence present in the dsz promoter DNA. Binding to the DNA also changes conformation of the protein.

Isolation and identification of a TetR family protein that regulates the biodesulfurization operon.

Biodesulfurization helps in removal of sulfur from organosulfur present in petroleum fractions. All microorganisms isolated to date harbor a desulfurization operon consisting of three genes dszA, -B and -C which encode for monooxygenases (DszA & C) and desulfinase (DszB). Most of the studies have been carried out using dibenzothiophene as the model organosulfur compound, which is converted into 2 hydroxybiphenyl by a 4S pathway which maintains the calorific value of fuel. There are few studies reported on the regulation of this operon. However, there are no reports on the proteins which can enhance the activity of the operon. In the present study, we used in vitro and in vivo methods to identify a novel TetR family transcriptional regulator from Gordonia sp. IITR100 which functions as an activator of the dsz operon. Activation by TetR family regulator resulted in enhanced levels of desulfurization enzymes in Gordonia sp. IITR100. Activation was observed only when the 385 bp full length promoter was used. Upstream sequences between - 385 and - 315 were found to be responsible for activation. We provide evidence that the TetR family transcription regulator serves as an activator in other biodesulfurizing microorganisms such as Rhodococcus erythropolis IGTS8 and heterologous host Escherichia coli. This is the first report on the isolation of a possible transcriptional regulator that activates the desulfurization operon resulting in improved biodesulfurization.

An improved method for the isolation and identification of unknown proteins that bind to known DNA sequences by affinity capture and mass spectrometry.

Transcription of a gene can be regulated at many different levels. One such fundamental level is interaction between protein and DNA. Protein(s) binds to distinct sites on the DNA, which activate, enhance or repress transcription. Despite being such an important process, very few tools exist to identify the proteins that interact with chromosome, most of which are in vitro in nature. Here, we propose an in vivo based method for identification of DNA binding protein(s) in bacteria where the DNA-protein complex formed in vivo is crosslinked by formaldehyde. This complex is further isolated and the bound proteins are identified. The method was used to isolate promoter DNA binding proteins, which bind and regulate the dsz operon in Gordonia sp. IITR 100 responsible for biodesulfurization of organosulfurs. The promoter binding proteins were identified by MALDI ToF MS/MS and the binding was confirmed by gel shift assay. Unlike other reported in vivo methods, this improved method does not require sequence of the whole genome or a chip and can be scaled up to improve yields.