Biochemical characterization of a novel thermostable chitinase from Hydrogenophilus hirschii strain KB-DZ44.

An extracellular acido-thermostable endo-chitinase (called ChiA-Hh59) from thermophilic Hydrogenophilus hirschii strain KB-DZ44, was purified and characterized. The maximum chitinase activity recorded after 36-h of incubation at 60°C was 3000U/ml. Pure enzyme was obtained after heat and acidic treatment, precipitation by ammonium sulphate and acetone, respectively, followed by sequential column chromatographies on Sephacryl S-200 and Mono Q-Sepharose. Based on Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 59103.12-Da. The 22 residue NH2-terminal sequence of the enzyme showed high homology with family-18 bacterial chitinases. The optimum pH and temperature values for chitinase activity were pH 5.0 and 85°C, respectively. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). The obtained results suggest that ChiA-Hh59 might be an endo-chitinase. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Its Km and kcat values were 0.298mg colloidal chitin/ml and 14400s-1, respectively. Its catalytic efficiency was higher than those of chitodextrinase and ChiA-65. Additionally, Thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Hh59 acted as an endo-splitting enzyme. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.

[THE EFFECT OF METAL IONES AND SPECIFIC CHEMICAL REAGENTS ON THE ACTIVITY OF ASPERGILLUS FLAVUS VAR. ORYZAE AND BACILLUS SUBTILIS α-AMYLASES].

The effect of cations and anions on the activity of Aspergillus flavus var. oryzae and Bacillus subtilis α-amylases showed that the tested enzymes are sensitive to most of cations and resistant to anions. The most significant inhibitory effects on the activity of A. flavus var. oryzae α-amylase have been demonstrated by Al3+ and Fe3+ ions, while on the activity of B. subtilis α-amylase - Hg2+, Cu2+ and Fe3+ ions. Inactivation of A. flavus var. oryzae and B. subtilis α-amylases in the presence of EGTA is indicated on the presence within their structure of metal ions. An important role in the enzymatic catalysis of both enzymes play carboxyl groups as evidenced by their inhibition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide. Inhibition of B. subtilis α-amylase by p-chloromercuribenzoate, N-ethylmaleimide and sodium sulfite is indicated on the probable involvement of the sulfhydryl groups in the functioning of the enzyme. Unlike most studied glycosidases the tested enzymes do not contain histidine imidazole group in the active center.

New strategy for reversible modulation of protein activity through site-specific conjugation of small molecule and polymer.

A new strategy for accurate and reversible modulation of protein activity via simple conjugation of the sulfhydryl modifier and polymer with the introduced Cys residue in protein was developed in this study. With Escherichia coli inorganic pyrophosphatase (PPase) as a model protein, we used site-directed mutagenesis to generate a mutant PPase (PPC) with a substituted Cys residue at the specific Lys-148 site, which is within a conserved sequence near the active site and exposed to the surface of the PPC for chemical reaction. The site-specific conjugation of the mutated Cys residue in PPC with sulfhydryl modifier p-chloromercuribenzoate (PCMB) and pyridyl disulfide-functionalized poly(2-hydroxyethyl methacrylate) (pHEMA) resulted in obvious decrease or complete loss of the catalytic activity of PPC, due to the conformational change of PPC. Compared with the effect of small molecule modification (PCMB), the pHEMA conjugation led to greater inhibitory effect on protein activity due to the significant change of the tertiary structure of PPC after conjugation. Moreover, the protein activity can be restored to different extents by the treatment with different amount of reductive reagents, which can result in the dissociation between PPC and PCMB or pHEMA to recover the protein conformation. This study provides a new strategy for efficient control of protein activity at different levels by site-specific conjugation of a small molecule and polymer.

Enhanced activity of an angiotensin-(1-7) neuropeptidase in glucocorticoid-induced fetal programming.

We previously identified angiotensin converting enzyme (ACE) and an endopeptidase activity that degraded angiotensin-(1-7) [Ang-(1-7)] to Ang-(1-5) and Ang-(1-4), respectively, in the cerebrospinal fluid (CSF) of 6-month old male sheep. The present study undertook a more comprehensive analysis of the CSF peptidase that converts Ang-(1-7) to Ang-(1-4) in control and in utero betamethasone-exposed sheep (BMX). Characterization of the Ang-(1-7) peptidase revealed that the thiol agents 4-aminophenylmercuric acetate (APMA) and p-chloromercuribenzoic acid (PCMB), as well as the metallo-chelators o-phenanthroline and EDTA essentially abolished the enzyme activity. Additional inhibitors for serine, aspartyl, and cysteine proteases, as well as selective inhibitors against the endopeptidases neprilysin, neurolysin, prolyl and thimet oligopeptidases did not attenuate enzymatic activity. Competition studies against the peptidase revealed similar IC50s for Ang-(1-7) (5µM) and Ang II (3µM), but lower values for Ala(1)-Ang-(1-7) and Ang-(2-7) of 1.8 and 2.0µM, respectively. In contrast, bradykinin exhibited a 6-fold higher IC50 (32µM) than Ang-(1-7) while neurotensin was a poor competitor. Mean arterial pressure (78±1 vs. 94±2mmHg, N=4-5, P<0.01) and Ang-(1-7) peptidase activity (14.2±1 vs 32±1.5fmol/min/ml CSF, N=5, P<0.01) were higher in the BMX group, and enzyme activity inversely correlated with Ang-(1-7) content in CSF. Lower Ang-(1-7) expression in brain is linked to baroreflex impairment in hypertension and aging, thus, increased activity of an Ang-(1-7) peptidase may contribute to lower CSF Ang-(1-7) levels, elevated blood pressure and impaired reflex function in this model of fetal programming.

An altered zinc-binding site confers resistance to a covalent inactivator of New Delhi metallo-beta-lactamase-1 (NDM-1) discovered by high-throughput screening.

Due to the global threat of antibiotic resistance mediated by New Delhi metallo-beta-lactamase-1 (NDM-1) and the lack of structurally diverse inhibitors reported for this enzyme, we developed screening and counter-screening assays for manual and automated formats. The manual assay is a trans-well absorbance-based endpoint assay in 96-well plates and has a Z' factor of 0.8. The automated assay is an epi-absorbance endpoint assay in 384-well plates, has a Z' factor of ≥0.8, good signal/baseline ratios (>3.8), and is likely scalable for high-throughput screening (HTS). A TEM-1-based counter-screen is also presented to eliminate false positives due to assay interference or off-target activities. A pilot screen of a pharmacologically characterized compound library identified two thiol-modifying compounds as authentic NDM-1 inhibitors: p-chloromecuribenzoate (p-CMB) and nitroprusside. Recombinant NDM-1 has one Cys residue that serves as a conserved active-site primary zinc ligand and is selectively modified by p-CMB as confirmed by LC-MS/MS. However a C208D mutation results in an enzyme that maintains almost full lactamase activity, yet is completely resistant to the inhibitor. These results predict that covalent targeting of the conserved active-site Cys residue may have drawbacks as a drug design strategy.

[Investigation of functional groups of Cryptococcus albidus alpha-L-rhamnosidase].

The effect of cations, anions and specific chemical reagents: 1-[3-(dimethylamino)propyl]-3-ethylcarbodimide methiodide, EDTA, o-phenantroline, dithiotreitol, L-cysteine, beta-mercaptoethanol, p-chlormercurybenzoate (p-ChMB), N-ethylmaleimide on the alpha-L-rhamnosidase activity of Cryptococcus albidus has been investigated. The essential role of Ag+ which inhibits the alpha-L-rhamnosidase activity by 72.5% was shown. Rhamnose at 1-5 mM protect the enzyme from the negative effect of Ag(+). It was expected that carboxyl group of C-terminal aminoacid and imidazole group of histidine would participate in the catalytic action of alpha-L-rhamnosidase on the basis of inhibition and kinetic analysis.

High-throughput assays probing protein-RNA interactions of eukaryotic translation initiation factors.

Protein-RNA interactions are involved in all facets of RNA biology. The identification of small molecules that selectively block such bimolecular interactions could provide insight into previously unexplored steps of gene regulation. Such is the case for regulation of eukaryotic protein synthesis where interactions between messenger RNA (mRNA) and several eukaryotic initiation factors govern the recruitment of 40S ribosomes (and associated factors) to mRNA templates during the initiation phase. We have designed simple fluorescence polarization-based high-throughput screening assays that query the binding of several translation factors to RNA and found that the mixed inhibitor p-chloromercuribenzoate interferes with poly(A) binding protein-RNA interaction.

Investigation of sulfhydryl groups in cabbage phospholipase D by combination of derivatization methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

All eight cysteine residues in 92 kDa cabbage phospholipase D (PLD), deduced from the cDNA sequence, were shown to have free sulfhydryl groups by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of tryptic peptides of PLD derivatized with p-chloromercurybenzoate, iodoacetic acid, and N-ethylmaleimide, as well as of underivatized PLD. Assignment of sulfhydryl groups by any one method was not conclusive. However, complementary information derived from tryptic peptides derivatized with different reagents made full assignment of sulfhydryl groups possible.

The role of actin sulfhydryls in actin-myosin interaction.

Rabbit white skeletal and dog cardiac actins combined fully with the heavy meromyosins at 4 degrees C and low ionic strength (10.03); the complexes (actin-heavy meromyosin) were mostly in a heavy component which settled rapidly during rotor acceleration. Increasing the temperature to 24 degrees C favored formation of the heavy component while increasing the ionic strength to 10.3 decreased formation of the heavy component. Modification of actin with p-mercuribenzoate (PCMB) generally reduced the heavy component and viscosity but did not interfere with actin-heavy meromyosin combination. Modification with iodoacetamide or N-ethylmaleimide had similar effects. Addition of ATP reduced viscosity and eliminated the heavy component at levels which partially dissociated actin-heavy meromyosin. Modification of heavy meromyosin with PCMB inhibited combination with actin. The characteristics of interaction of the cardiac proteins varied less with temperature but more with ionic strength than those of skeletal proteins.