Dynamic elements govern the catalytic activity of CapE,a capsular polysaccharide-synthesizing enzyme from Staphylococcus aureus

CapE is an essential enzyme for the synthesis of capsular polysaccharide (CP) of pathogenic strains of Staphylococcus aureus. Herein we demonstrate that CapE is a 5-inverting 4,6-dehydratase enzyme. However, in the absence of downstream enzymes, CapE catalyzes an additional reaction (5-back-epimerization) affording a by-product under thermodynamic control. Single-crystal X-ray crystallography was employed to identify the structure of the by-product. The structural analysis reveals a network of coordinated motions away from the active site governing the enzymatic activity of CapE. A second dynamic element (the latch) regulates the enzymatic chemoselectivity. The validity of these mechanisms was evaluated by site-directed mutagenesis.     

Characterization of the type 2 NADH:menaquinone oxidoreductases from Staphylococcus aureus and the bactericidal action of phenothiazines

Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple drug resistant bacterial pathogens causing serious infections, many of which are life-threatening. Consequently, new therapeutic targets are required to combat such infections. In the current work, we explore the type 2 Nicotinamide adenine dinucleotide reduced form (NADH) dehydrogenases (NDH-2s) as possible drug targets and look at the effects of phenothiazines, known to inhibit NDH-2 from Mycobacterium tuberculosis. NDH-2s are monotopic membrane proteins that catalyze the transfer of electrons from NADH via flavin adenine dinucleotide (FAD) to the quinone pool. They are required for maintaining the NADH/Nicotinamide adenine dinucleotide (NAD⁺) redox balance and contribute indirectly to the generation of proton motive force. NDH-2s are not present in mammals, but are the only form of respiratory NADH dehydrogenase in several pathogens, including S. aureus. In this work, the two putative ndh genes present in the S. aureus genome were identified, cloned and expressed, and the proteins were purified and characterized. Phenothiazines were shown to inhibit both of the S. aureus NDH-2s with half maximal inhibitory concentration (IC₅₀) values as low as 8 μM. However, evaluating the effects of phenothiazines on whole cells of S. aureus was complicated by the fact that they are also acting as uncouplers of oxidative phosphorylation. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Bacteriophage enzymes for the prevention and treatment of bacterial infections: Stability and stabilization of the enzyme lysing <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> cells

The effect of various compounds on the activity and stability of a phage-associated enzyme lysing cells of streptococci of groups A and C (PlyC) was investigated. Substantial inhibition of the enzyme activity was revealed at an increased ionic strength (in the presence of NaCl) and upon the addition of carbohydrates (mono-, di-, and polysaccharides), i.e., agents stabilizing many enzymes. It was established that the enzyme activity was substantially reduced in the presence of positively charged polyelectrolytes and surfactants, whereas incubation with micelle-forming substances and negatively charged polyelectrolytes led to PlyC activation and stabilization. It was shown that, in the micellar polyelectrolyte composition M16, the enzyme retained its activity for 2 months; while in a buffer solution under the same conditions (pH 6.3, room temperature), ture), it practically completely lost its activity in 2 days. Characteristics of the enzyme thermal inactivation were found, in particular, its half-inactivation time at various temperatures; these allowed us to estimate its behavior at any temperature and to recommend conditions for its storage and use.     

Design of β‐galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance

The use of heterogeneous biocatalysis in industrial applications is advantageous and the enzyme stability improvement is a continuous challenge. Therefore, we designed β‐galactosidase heterogeneous biocatalysts by immobilization, involving the support synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis, and Aspergillus oryzae). The underivatized, tailored, macro‐mesoporous silica exhibited high surface area, offered high enzyme immobilization yields and activity. Its chemical activation with glyoxyl groups bound the enzyme covalently, which suppressed lixiviation and conferred higher pH and thermal stability (120‐fold than for the soluble enzyme), without observable reduction of activity/stability due to the presence of silica. The best balance between the immobilization yield (68%), activity (48%), and stability was achieved for Bacillus circulans β‐galactosidase immobilized on glyoxyl‐activated silica, without using stabilizing agents or modifying the enzyme. The enzyme stabilization after immobilization in glyoxyl‐activated silica was similar to that observed in macroporous agarose‐glyoxyl support, with the reported microbiological and mechanical advantages of inorganic supports. The whey lactolysis at pH 6.0 and 25°C by using this catalyst (1 mg ml^?1, 290 UI g^?1) was still 90%, even after 10 cycles of 10 min, in batch process but it could be also implemented on continuous processes at industrial level with similar results.     

Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester

Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S–O bond cleavage) or carbon (C–O bond cleavage). In primary and secondary alkyl sulfates, attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars, attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases (ASs) catalyze S–O cleavage in sulfate sugars and arylsulfates, and alkyl sulfatases break the C–O bond of alkyl sulfates. Sinorhizobium meliloti choline sulfatase (SmCS) efficiently catalyzes the hydrolysis of alkyl sulfate choline-O-sulfate (k_cat/K_M = 4.8 × 10³ s^? 1 M^? 1) as well as arylsulfate 4-nitrophenyl sulfate (k_cat/K_M = 12 s^? 1 M^? 1). Its 2.8-Å resolution X-ray structure shows a buried, largely hydrophobic active site in which a conserved glutamate (Glu386) plays a role in recognition of the quaternary ammonium group of the choline substrate. SmCS structurally resembles members of the alkaline phosphatase superfamily, being most closely related to dimeric ASs and tetrameric phosphonate monoester hydrolases. Although > 70% of the amino acids between protomers align structurally (RMSDs 1.79–1.99 Å), the oligomeric structures show distinctly different packing and protomer–protomer interfaces. The latter also play an important role in active site formation. Mutagenesis of the conserved active site residues typical for ASs, H₂¹⁸O-labeling studies and the observation of catalytically promiscuous behavior toward phosphoesters confirm the close relation to alkaline phosphatase superfamily members and suggest that SmCS is an AS that catalyzes S–O cleavage in alkyl sulfate esters with extreme catalytic proficiency.     

Development of a bioassay from isolated digestive gland cells of the cuttlefish Sepia officinalis L. (Mollusca Cephalopoda): effect of Cu, Zn and Ag on enzyme activities and cell viability

The purpose of this study was to establish a bioassay from isolated digestive gland cells of the cuttlefish Sepia officinalis in order to observe the effect of heavy metals on digestive enzyme activities. Digestive cells were isolated using a pronase enzyme that was removed by several washings of the cell suspension. Cell viability was tested by the MTT assay (3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium) and microscopic analysis. The results showed that isolated digestive cells could be maintained 24 h with preservation of whole digestive functionality, measured in terms of MTT test. In fact, the viability was maintained at a high level during 24 h and the intra- and extracellular digestive enzyme activities became stabilised rapidly. Furthermore, suspension cells responded to calcium ionophore and 8-Bromo-cAMP by an unspecific secretion of extracellular digestive enzyme, trypsin, which demonstrated that isolated digestive cells were functional. Using the bioassay, ecotoxicological studies showed that heavy metals could have effects on digestive enzyme activities after 24 h of an incubation time of the metal with the cells. In fact, zinc and silver affected trypsin and/or cathepsins specific activity of the cells. On the contrary, copper had no effect on digestive enzyme activities. Zinc, which is a trace element in all living animals, generated two different responses of cathepsins and cell viability. At a low concentration (0.02 μM), it increased viability and cathepsins specific activity, whereas at a high concentration (0.02 mM), zinc inhibited the cathepsins specific activity with an inhibition of cathepsins. For silver, whatever the tested concentration (0.02 mM or 0.02 μM), it has no impact on digestive gland isolated cell viability. Nevertheless, heavy metal induced high disturbance of enzymatic systems.     

Pyruvate carboxylase from Mycobacterium smegmatis: stabilization,rapid purification,molecular and biochemical characterization and regulation of the cellular level

This is the first report on the purification and characterization of an anaplerotic enzyme from a Mycobacterium. The anaplerotic reactions play important roles in the biochemical differentiation of mycobacteria into non-replicating stages. We have purified and characterized a pyruvate carboxylase (PYC) from Mycobacterium smegmatis and cloned and sequenced its gene. We have developed a very rapid and efficient purification protocol that provided PYC with very high specific activities (up to 150 U/mg) that remained essentially unchanged over a month. The enzyme was found to be a homomultimer of 121 kDa subunits, mildly thermophilic, absolutely dependent on acyl-CoAs for activity and inhibited by ADP, by excess Mg²⁺, Co²⁺, and Mn²⁺, by aspartate, but not by glutamate and α-ketoglutarate. Supplementation of minimal growth medium with aspartate did not lower the cellular PYC level, rather doubled it; with glutamate the level remained unchanged. These observations would not fit the idea that the M. smegmatis enzyme fulfills a straightforward anaplerotic function; in a closely related organism, Corynebacterium glutamicum, PYC is the major anaplerotic enzyme. Growth on glucose provided 2-fold higher cellular PYC level than that observed with glycerol. The PYCs of M. smegmatis and Mycobacterium tuberculosis were highly homologous to each other. In M. smegmatis, M. tuberculosis and M. lepra, pyc was flanked by a putative methylase and a putative integral membrane protein genes in an identical operon-like arrangement. Thus, M. smegmatis could serve as a model for studying PYC-related physiological aspects of mycobacteria. Also, the ease of purification and the extraordinary stability could make the M. smegmatis enzyme a model for studying the structure–function relationships of PYCs in general. It should be noted that no crystal structure is available for this enzyme of paramount importance in all three domains of life, archaea, bacteria, and eukarya.     

Cytochrome P450 1A-dependent enzyme activities in the liver of dab (Limanda limanda): kinetics, seasonal changes and detection limits

Concentrations of total cytochrome P450 and cytochrome P450 1A (CYP 1A) and activities of ethoxycoumarin O-deethylase (ECOD), ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) were measured in the liver of prespawning, spawning and postspawning dab (Limanda limanda) from the German Bight. Between all P450-dependent parameters measured significant correlations were found. Generally, during prespawning and spawning season higher values were measured in the liver of males compared to females, but the ratio between sexes changed during spawning time, when concentrations and activities in the liver of males decreased and increased in the liver of females. The activity and the signal-to-noise ratio decrease in the order EROD, ECOD and PROD. This decrease is accompanied by an increase in K_m. The findings indicate that the different activities can be attributed to the strongly overlapping substrate specificity and the different enzyme affinities of one enzyme, CYP 1A, towards the three substrates. A biphasic kinetic of ECOD indicates that in addition to CYP 1A a second isozyme catalyses the O-deethylation of ethoxycoumarin in the liver of dab. Interestingly, the ratio between EROD activity and CYP 1A concentration varied seasonally but did not differ significantly between sexes.     

Development and ultrastructure of a microsporidian parasite in midgut cells of the larch sawfly, Pristiphora erichsonii (Hymenoptera: Tenthredinidae)

The developmental stages of a microsporidium from larvae of Pristiphora erichsonii were investigated. Meronts appeared to be the only stage containing a diplokaryon. Nuclei of sporonts in a parasitophorous vesicle underwent at least three divisions and uninucleate sporoblasts developed from these multinucleate sporonts. As many as 38 spores were observed with a vesicle. A thin pansporoblastic membrane limited the vesicle and was derived from rough endoplasmic reticulum of the host cell. The microsporidium was tentatively identified as Pleistophora sp. Infection levels of the microsporidium in natural populations of P. erichsonii reached 25%.     

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla,TriVal, and TriLeu variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.