Sulphonamide inhibition studies of the ß-carbonic anhydrase from the bacterial pathogen Clostridium perfringens.

The ß-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Clostridium perfringens (CpeCA) was recently characterised kinetically and for its anion inhibition profile. In the search of effective CpeCA inhibitors, possibly useful to inhibit the growth/pathogenicity of this bacterium, we report here an inhibition study of this enzyme with a panel of aromatic, heterocyclic and sugar sulphonamides/sulphamates. Some sulphonamides, such as acetazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, sulthiame and 4-(2-hydroxymethyl-4-nitrophenyl-sulphonamido)ethylbenzenesulphonamide were effective CpeCA inhibitors, with KIs in the range of 37.4-71.6 nM. Zonisamide and saccharin were the least effective such inhibitors, whereas many other aromatic and heterocyclic sulphonamides were moderate - weak inhibitors with KIs ranging between 113 and 8755 nM. Thus, this study provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.

Structural and Biochemical Characterizations of Methanoredoxin from Methanosarcina acetivorans, a Glutaredoxin-Like Enzyme with Coenzyme M-Dependent Protein Disulfide Reductase Activity.

Glutaredoxins (GRXs) are thiol-disulfide oxidoreductases abundant in prokaryotes, although little is understood of these enzymes from the domain Archaea. The numerous characterized GRXs from the domain Bacteria utilize a diversity of low-molecular-weight thiols in addition to glutathione as reductants. We report here the biochemical and structural properties of a GRX-like protein named methanoredoxin (MRX) from Methanosarcina acetivorans of the domain Archaea. MRX utilizes coenzyme M (CoMSH) as reductant for insulin disulfide reductase activity, which adds to the diversity of thiol protectants in prokaryotes. Cell-free extracts of M. acetivorans displayed CoMS-SCoM reductase activity that complements the CoMSH-dependent activity of MRX. The crystal structure exhibits a classic thioredoxin-glutaredoxin fold comprising three α-helices surrounding four antiparallel ß-sheets. A pocket on the surface contains a CVWC motif, identifying the active site with architecture similar to GRXs. Although it is a monomer in solution, the crystal lattice has four monomers in a dimer of dimers arrangement. A cadmium ion is found within the active site of each monomer. Two such ions stabilize the N-terminal tails and dimer interfaces. Our modeling studies indicate that CoMSH and glutathione (GSH) bind to the active site of MRX similar to the binding of GSH in GRXs, although there are differences in the amino acid composition of the binding motifs. The results, combined with our bioinformatic analyses, show that MRX represents a class of GRX-like enzymes present in a diversity of methane-producing Archaea.

Structural and Biochemical Characterization of a Ferredoxin:Thioredoxin Reductase-like Enzyme from Methanosarcina acetivorans.

Bioinformatics analyses predict the distribution in nature of several classes of diverse disulfide reductases that evolved from an ancestral plant-type ferredoxin:thioredoxin reductase (FTR) catalytic subunit to meet a variety of ecological needs. Methanosarcina acetivorans is a methane-producing species from the domain Archaea predicted to encode an FTR-like enzyme with two domains, one resembling the FTR catalytic subunit and the other containing a rubredoxin-like domain replacing the variable subunit of present-day FTR enzymes. M. acetivorans is of special interest as it was recently proposed to have evolved at the time of the end-Permian extinction and to be largely responsible for the most severe biotic crisis in the fossil record by converting acetate to methane. The crystal structure and biochemical characteristics were determined for the FTR-like enzyme from M. acetivorans, here named FDR (ferredoxin disulfide reductase). The results support a role for the rubredoxin-like center of FDR in transfer of electrons from ferredoxin to the active-site [Fe4S4] cluster adjacent to a pair of redox-active cysteines participating in reduction of disulfide substrates. A mechanism is proposed for disulfide reduction similar to one of two mechanisms previously proposed for the plant-type FTR. Overall, the results advance the biochemical and evolutionary understanding of the FTR-like family of enzymes and the conversion of acetate to methane that is an essential link in the global carbon cycle and presently accounts for most of this greenhouse gas that is biologically generated.

Prokaryotic carbonic anhydrases of Earth's environment.

Carbonic anhydrase is a metalloenzyme catalyzing the reversible hydration of carbon dioxide to bicarbonate. Five independently evolved classes have been described for which one or more are found in nearly every cell type underscoring the general importance of this ubiquitous enzyme in Nature. The bulk of research to date has centered on the enzymes from mammals and plants with less emphasis on prokaryotes. Prokaryotic carbonic anhydrases play important roles in the ecology of Earth's biosphere including acquisition of CO2 for photosynthesis and the physiology of aerobic and anaerobic prokaryotes decomposing the photosynthate back to CO2 thereby closing the global carbon cycle. This review focuses on the physiology and biochemistry of carbonic anhydrases from prokaryotes belonging to the domains Bacteria and Archaea that play key roles in the ecology of Earth's biosphere.

Biochemistry and physiology of the ß class carbonic anhydrase (Cpb) from Clostridium perfringens strain 13.

The carbonic anhydrase (Cpb) from Clostridium perfringens strain 13, the only carbonic anhydrase encoded in the genome, was characterized both biochemically and physiologically. Heterologously produced and purified Cpb was shown to belong to the type I subclass of the ß class, the first ß class enzyme investigated from a strictly anaerobic species of the domain Bacteria. Kinetic analyses revealed a two-step, ping-pong, zinc-hydroxide mechanism of catalysis with Km and kcat/Km values of 3.1 mM CO2 and 4.8 × 106 s⁻¹ M⁻¹, respectively. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semidefined medium and an atmosphere without CO2. The growth of the mutant was the same as that of the parent wild-type strain when cultured in nutrient-rich media with or without CO2 in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO2 fixation reactions by supplying bicarbonate to carboxylases. Potential roles in competitive fitness are discussed.

Anion inhibition studies of a ß-carbonic anhydrase from Clostridium perfringens.

A ß-carbonic anhydrases (CAs, EC 4.2.1.1) was recently cloned, purified and characterized kinetically in the pathogen Clostridium perfringens. We report here the first inhibition study of this enzyme (CpeCA). CpeCA was poorly inhibited by iodide and bromide, and was inhibited with KIs in the range of 1-10mM by a range of anions such as (thio)cyanate, azide, bicarbonate, nitrate, nitrite, hydrogensulfite, hydrogensulfide, stannate, tellurate, pyrophosphate, divanadate, tetraborate, peroxydisulfate, sulfate, iminodisulfonate and fluorosulfonate. Better inhibitory power, with K(I)s of 0.36-1.0 mM, was observed for cyanide, carbonate, selenate, selenocyanide, trithiocarbonate and diethyldithiocarbamate, whereas the best CpeCA inhibitors were sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, which had KIs in the range of 7-75 µM. This study thus provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.

Are We Justified Doing Salvage or Amputation Procedure Based on Mangled Extremity Severity Score in Mangled Upper Extremity Injury.

INTRODUCTION: Mangled upper limb injuries are at surge because of industrialization, modernization, and severe motor vehicle accidents. The utility of various scoring systems are meant for decision making in mangled lower limb injuries, and the same have been extrapolated for mangled upper limb injuries to make a decision of salvage or amputation for the lack of separate scoring system. MATERIALS AND METHODS: We applied mangled extremity severity score (MESS) and mangled extremity syndrome index (MESI) scoring systems to 10 cases of mangled upper limb injuries during the period of November 2010 to September 2012 presented at our tertiary trauma care center. Average MESS score was 7.7 and MESI score was 18.1. Above elbow, amputation was needed in three patients, and salvage procedure was done in rest of the seven patients. All the patients were subjected to salvage procedure initially unless life threatening because of mangled limb injury. MESS scores over 7, MESI score over 20 is accepted for amputation in lower limbs, but could not be justified in our study for MESS whereas MESI was more reliable. Functional outcome was assessed using visual analog scale score and short form-36 (SF-36) score for all patients, which was satisfactory, elderly and diabetic patients were relatively less satisfied. CONCLUSIONS: (a) Upper limb and lower limb mangled injuries cannot be considered same because of their anatomy. The upper limb has more rich vascularity and efficient collaterals, small muscle mass, so ischemia time is relatively more. Therefore, the different scoring systems are needed for both. (b) In our case series, MESI scoring was more reliable then MESS score, but this needs a large prospective study to validate it. (c) Salvage should be prime realistic aim influenced by several factors. Prosthesis for upper limb is very expensive and not well tolerated, so even a woody limb is well accepted in our patients.

Case Report of Two Cases of Patella Subacute Osteomyelitis in Diabetic Women A Rare Entity.

INTRODUCTION: Patella osteomyelitis is a rare entity in adults. Most often it is seen in children of five to twelve years of age because of its unique ossification and vascularity. Immuno compromised states like HIV, tuberculosis, intravenous drug abuse and trauma have been predisposing factors for adult patellar osteomyelitis. We report two cases of patellar osteomyelitis in adult diabetic women with uncontrolled glycemic levels and having no previous history of any trauma or systemic infection. CASE REPORT: A 43-year-old diabetic woman presented with complaints of left knee pain and swelling with no history of trauma. On examination, pointed tenderness was present over anterior aspect of patella with patellar grind test positive. Radiography and MRI revealed solitary well circumscribed patellar cyst. Lateral chondral blisters were noted while doing arthroscopy and secretions oozed out on puncturing. Curettage was carried out for the same. Culture and sensitivity revealed no growth and the patient was prescribed antibiotics for 6 weeks. Second case was a 46-year-old diabetic lady with similar presentation. MRI additionally showed abscess in intermuscular plains around knee joint. An aspirated fluid was negative for growth of organisms. Knee arthrotomy and curettage of patellar sinus tracts was done with evacuation of intramuscular abscess. Antibiotics were given for 6 weeks. Both patients had complete relief of symptoms. CONCLUSION: Patella osteomyelitis in adults is very rare. In patients with uncontrolled diabetes, vague anterior knee pain, elevated ESR and CRP, one should keep patellar subacute osteomyelitis as a differential diagnosis which can be further confirmed by X-ray, MRI or bone scan. An appropriate early treatment with antibiotics and surgical intervention can give a satisfactory result.

Thermal stability of alpha-amylase from malted jowar (Sorghum bicolor).

Malted cereals are rich sources of alpha-amylase, which catalyzes the random hydrolysis of internal alpha-(1-4)-glycosidic bonds of starch, leading to liquefaction. Amylases play a role in the predigestion of starch, leading to a reduction in the water absorption capacity of the cereal. Among the three cereal amylases (barley, ragi, and jowar), jowar amylase is found to be the most thermostable. The major amylase from malted jowar, a 47 kDa alpha-amylase, purified to homogeneity, is rich in beta structure ( approximately 60%) like other cereal amylases. T(m), the midpoint of thermal inactivation, is found to be 69.6 +/- 0.3 degrees C. Thermal inactivation is found to follow first-order kinetics at pH 4.8, the pH optimum of the enzyme. Activation energy, E(a), is found to be 45.3 +/- 0.2 kcal mol(-)(1). The activation enthalpy (DeltaH), entropy (DeltaS*), and free energy change (DeltaG) are calculated to be 44.6 +/- 0.2 kcal mol(-)(1), 57.1 +/- 0.3 cal mol(-)(1) K(-)(1), and 25.2 +/- 0.2 kcal mol(-)(1), respectively. The thermal stability of the enzyme in the presence of the commonly used food additives NaCl and sucrose has been studied. T(m) is found to decrease to 66.3 +/- 0.3, 58.1 +/- 0.2, and 48.1 +/- 0.5 degrees C, corresponding to the presence of 0.1, 0.5, and 1 M NaCl, respectively. Sucrose acts as a stabilizer; the T(m) value is found to be 77.3 +/- 0.3 degrees C compared to 69.6 +/- 0.3 degrees C in the control.

Conformational stability of alpha-amylase from malted sorghum (Sorghum bicolor): reversible unfolding by denaturants.

Alpha-amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50mM Hepes containing 13.6mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 degrees C is characterized by two state transition with DeltaG (H(2)O) of 16.5 kJ mol(-1) and 22 kJ mol(-1), respectively, at pH 4.8 and pH 7.0 for GuHCl and DeltaG (H(2)O) of 25.2 kJ mol(-1) at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (DeltaC(p)), the unfolding enthalpy (H(g)) and the temperature at DeltaG=0 (T(g)) are 17.9+/-0.7 kJ mol(-1) K(-1), 501.2+/-18.2 kJ mol(-1) and 337.3+/-6.9 K at pH 4.8 and 14.3+/-0.5 kJ mol(-1) K(-1), 509.3+/-21.7 kJ mol(-1) and 345.4+/-4.8K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the 'B' domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by DeltaG (H(2)O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.