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.

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.