Chaperone function in organic co-solvents: experimental characterization and modeling of a hyperthermophilic chaperone subunit from Methanocaldococcus jannaschii

Molecular chaperones play a central role in maintaining protein structure within a cell. Previously, we determined that the gene encoding a molecular chaperone, a thermosome, from the hyperthermophilic archaeon Methanocaldococcus jannaschii is upregulated upon lethal heat shock. We have recombinantly expressed this thermosome (rTHS) and show here that it is both stable and fully functional in aqueous solutions containing water-miscible organic co-solvents. Based on circular dichroism the secondary structure of rTHS was not affected by one-hour exposures to a variety of co-solvents including 30% v/v acetonitrile (ACN) and 50% methanol (MeOH). By contrast, the secondary structure of a mesophilic homologue, GroEL/GroES (GroE), was substantially disrupted. rTHS reduced the aggregation of ovalbumin and citrate synthase in 30% ACN, assisted refolding of citrate synthase upon solvent-inactivation, and stabilized citrate synthase and glutamate dehydrogenase in the direct presence of co-solvents. Apparent total turnover numbers of these enzymes in denaturing solutions increased by up to 2.5-fold in the presence of rTHS. Mechanistic models are proposed to help ascertain specific conditions that could enhance or limit organic solvent-induced chaperone activity. These models suggest that thermodynamic stability and the reversibility of enzyme unfolding play key roles in the effectiveness of enzyme recovery by rTHS.     

Identification of a critical chaperoning region on an archaeal recombinant thermosome

Chaperone function in water-miscible organic co-solvents is useful for biocatalytic applications requiring enzyme stability in semi-aqueous media and for understanding chaperone behavior in hydrophobic environments. Previously, we have shown that a recombinant single subunit thermosome (rTHS) from Methanocaldococcus jannaschii functions in multiple co-solvents to hydrolyze ATP, prevent protein aggregation, and refold enzymes following solvent denaturation. For the present study, a truncated analog to the thermosome in which 70 N-terminal amino acids are removed is used to identify important regions within the thermosome for its chaperoning functions in organic co-solvents. Data presented herein indicate that the N-terminal region of rTHS is essential for the chaperone to restore the native state of the enzyme citrate synthase, but it is not a critical region for either binding of unfolded proteins or ATP hydrolysis. This is the first demonstration that direct refolding by a Group II chaperonin requires the N-terminal region of the protein.     

Small molecule inhibition of a Group II chaperonin: pinpointing a loop region within the equatorial domain as necessary for protein refolding

The functionality of regions within the equatorial domain of Group II chaperonins is poorly understood. Previously we showed that a 70 amino acid sequence within this domain on the single-subunit recombinant thermosome from Methanocaldococcus jannaschii (rTHS) contains residues directly responsible for refolding protein substrates [L.M. Bergeron, C. Lee, D.S. Clark, Identification of a critical chaperoning region on an archaeal recombinant thermosome, Biochem. Biophys. Res. Commun. 369 (2008) 707-711]. In the present study, 6-aminopenicillanic acid (6-APA) was found to bind to rTHS and inhibit it from refolding proteins. Fluorescence anisotropy was used to measure a 6-APA/rTHS dissociation constant of 17.1 μM and verify that the binding site is within the first 70 amino-terminal rTHS residues. Docking simulations point to a specific loop region at residues 53-57 on rTHS as the most likely binding region. This loop region is located within the oligomeric association sites of the wild-type thermosome. These results implicate a specific equatorial region of Group II chaperonins in the refolding of proteins, and suggest its importance in conformational changes that accompany chaperone function.     

Methylation in methanol-water mixtures: the effect of solvent composition and high pressure

The effect of pressure (0.1 to 400 MPa) and solvent composition (methanol concentration of 5 to 30%) on the synthesis of beta-methylgalactoside was studied. Galactose was used as a reactant and the reaction was catalyzed by beta-galactosidase from Aspergillus oryzae. Under the applied conditions the enzyme was sufficiently stable and the reaction equilibrium was reached. Higher methanol concentrations obviously influenced the product yield positively due to an increase in reactant concentration but also due to a solvent effect. This solvent effect can be explained by measurement of the activities of galactose and methylgalactoside. These results may be generalized to other methylations in methanol-water systems, where methanol positively affects synthesis yields. Pressure had a small, negative effect on synthesis yields.     

Ester synthesis in aqueous media in the presence of various lipases

Summary The ability of seven lipase preparations to catalyse methyl ester synthesis in aqueous media was compared and the synthesis reaction (esterification or alcoholysis) determined. Three behaviours were observed: three enzymes catalysed ester synthesis by esterification of free fatty acids and one enzyme catalysed alcoholysis but the other three lipases did not catalyse a net ester synthesis under the conditions tested. The three groups also differed by the influence of methanol on the hydrolysis reaction. The first group was not significantly inhibited up to the highest methanol concentration tested (5 M). Hydrolysis in the presence of the enzyme of the second group was increasingly inhibited with increasing methanol concentrations. In the presence of the third group, hydrolysis was 40 to 50% inhibited for all the concentrations tested (0.2–5 M).     

Enzyme reaction engineering: effect of methanol on the synthesis of antibiotics catalyzed by immobilized penicillin G acylase under isothermal and non-isothermal conditions

The effect of methanol on the kinetically controlled synthesis of cephalexin by free and immobilized penicillin G acylase (PGA) was investigated. Catalytic and hydrophobic membranes were obtained by chemical grafting, activation, and PGA immobilization on hydrophobic nylon supports. Butyl methacrylate (BMA) was used as graft monomer. Increasing concentrations of methanol were found to cause a greater deleterious effect on the activity of free than on that of the immobilized enzyme. Methanol, however, improved the kinetic stability of cephalexin synthesized by free PGA, resulting in higher maximum yields. By contrast, immobilized PGA reached 100% yields even in the absence of the cosolvent. Cephalexin synthesis by the catalytic membrane was also performed in a non-isothermal bioreactor. Under these conditions, a 94% increase of the synthetic activity and complete conversion of the limiting substrate to cephalexin were obtained. The addition of methanol reduced the non-isothermal activity increase. The physical cause responsible for the non-isothermal behavior of the hydrophobic catalytic membrane was identified in the process of thermodialysis.     

Regulation of methanol metabolism in the facultative methylotroph Nocardia sp. 239 during growth on mixed substrates in batch- and continuous cultures

The regulation of methanol metabolism in Nocardia sp. 239 was investigated. Growth on mixtures of glucose or acetate plus methanol in batch cultures resulted in simultaneous utilization of the substrates. The presence of glucose, but not of acetate, repressed synthesis of the ribulose monophosphate (RuMP) cycle enzymes hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), and methanol was used as an energy source only. Comparable results were obtained following addition of formaldehyde (fed-batch system) to a culture growing on glucose. The synthesis of the methanol dissimilatory and assimilatory enzymes in Nocardia sp. 239 thus appears to be controlled differently. Methanol and/or formaldehyde induce the synthesis of these enzymes, but under carbon-excess conditions their inducing effect on HPS and HPI synthesis is completely overruled by glucose, or metabolites derived from it. Repression of the synthesis of these RuMP cycle enzymes was of minor importance under carbon- and energy-limiting conditions in chemostat cultures. Addition of a pulse of glucose to a formaldehyde-limited (2.5 mmol l^–1 h^–1) fed-batch culture resulted in a decrease in the levels of several enzymes of methanol metabolism (including HPI), whereas the HPS levels remained relatively constant. Increasing HPS/HPI activity ratios were also observed with increasing growth rates in formaldehyde-limited chemostat cultures. The data indicate that additional mechanisms, the identity of which remains to be elucidated, are involved in controlling the levels of these C₁-specific enzymes in Nocardia sp. 239.Abbreviations HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - RuMP ribulose monophosphate - FBP fructose-1,6-bisphosphate - PFK 6-phosphofructokinase     

L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degrees C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degrees C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.     

Changes in conformation, stability and condensation of DNA by univalent and divalent cations in methanol-water mixtures

Circular dichroism spectroscopy, absorption spectroscopy, measurements of Tm values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na+ or Cs+) concentration and also in the presence of divalent cations Ca2+, Mg2+, and Mn2+. In the absence of divalent cations only slight conformational changes occurred and no condensation and/or aggregation could be detected. The Tm values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs+ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occurred and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.     

Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol supports

The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol-disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol-disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12-15-fold comparing optimal derivatives with their just-thiol immobilized counterparts.     

Poly-gamma-glutamate in bacteria

Poly-gamma-glutamate (PGA), a natural polymer, is synthesized by several bacteria (all Gram-positive), one archaea and one eukaryote. PGA has diverse biochemical properties, enabling it to play different roles, depending on the organism and its environment. Indeed, PGA allows bacteria to survive at high salt concentrations and may also be involved in virulence. The minimal gene sets required for PGA synthesis were recently defined. There are currently two nomenclatures depending on the PGA final status: cap, for 'capsule', when PGA is surface associated or pgs, for 'polyglutamate synthase', when PGA is released. The minimal gene sets contain four genes termed cap or pgs B, C, A and E. The PGA synthesis complex is membrane-anchored and uses glutamate and ATP as substrates. Schematically, the reaction may be divided into two steps, PGA synthesis and PGA transport through the membrane. PGA synthesis depends primarily on CapB-CapC (or PgsB-PgsC), whereas PGA transport requires the presence, or the addition, of CapA-CapE (or PgsAA-PgsE). The synthesis complex is probably responsible for the stereochemical specificity of PGA composition. Finally, PGA may be anchored to the bacterial surface or released. An additional enzyme is involved in this reaction: either CapD, a gamma-glutamyl-transpeptidase that catalyses anchorage of the PGA, or PgsS, a hydrolase that facilitates release. The anchoring of PGA to the bacterial surface is important for virulence. All cap genes are therefore potential targets for inhibitors specifically blocking PGA synthesis or anchorage.     

Sulfonate protecting groups. Regioselective sulfonylation of myo-inositol orthoesters-improved synthesis of precursors of D- and L-myo-inositol 1,3,4,5-tetrakisphosphate,myo-inositol 1,3,4,5,6-pentakisphosphate and related derivatives

The regioselectivity of sulfonylation of myo-inositol orthoesters was controlled by the use of different bases to obtain the desired sulfonate. Monosulfonylation of myo-inositol orthoesters in the presence of one equivalent of sodium hydride or triethylamine resulted in the sulfonylation of the 4-hydroxyl group. The use of pyridine as a base for the same reaction resulted in sulfonylation of the 2-hydroxyl group. Disulfonylation of these orthoesters in the presence of excess sodium hydride yielded the 4,6-di-O-sulfonylated orthoesters. However, the use of triethylamine or pyridine instead of sodium hydride yielded the 2,4-di-O-sulfonylated orthoester. Sulfonylated derivatives of myo-inositol orthoesters were stable to conditions of O-alkylation but were cleaved using magnesium/methanol or sodium methoxide in methanol to regenerate the corresponding myo-inositol orthoester derivative. These new methods of protection-deprotection have been used: (i) for the efficient synthesis of enantiomers of 2,4-di-O-benzyl-myo-inositol, which are precursors for the synthesis of D- and L-myo-inositol 1,3,4,5-tetrakisphosphate; (ii) for the preparation of 2-O-benzyl-myo-inositol which is a precursor for the preparation of myo-inositol 1,3,4,5,6-pentakisphosphate.     

Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis.

An enzymatic system for poly gamma-glutamate (PGA) synthesis in Bacillus subtilis, the PgsBCA system, was investigated. The gene-disruption experiment showed that the enzymatic system was the sole machinery of PGA synthesis in B. subtilis. We succeeded in achieving the enzymatic synthesis of elongated PGAs with the cell membrane of the Escherichia coli clone producing PgsBCA in the presence of ATP and D-glutamate. The enzyme preparation solubilized from the membrane with 8 mM Chaps catalyzed ADP-forming ATP hydrolysis only in the presence of glutamate; the D-enantiomer was the best cosubstrate, followed by the L-enantiomer. Each component of the system, PgsB, PgsC, and PgsA, was translated in vitro and the glutamate-dependent ATPase reaction was kinetically analyzed. The PGA synthetase complex, PgsBCA, was suggested to be an atypical amide ligase.     

Changes in Conformation,Stability and Condensation of DNA by Univalent and Divalent Cations in Methanol-Water Mixtures

Abstract

Circular dichroism spectroscopy, absorption spectroscopy, measurements of T_m values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na⁺ or Cs⁺) concentration and also in the presence of divalent cations Ca²⁺, Mg²⁺, and Mn²⁺. In the absence of divalent cations only slight conformational changes occured and no condensation and/or aggregation could be detected. The T_m values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs⁺ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occured and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.     

Serine synthesis in cyanobacteria by a nonphotorespiratory pathway

The pathway of serine synthesis was investigated in six species of cyanobacteria: Anabaena cylindrica, A. variabilis, A. flos-aquae, Coccochloris peniocystis, Gleo-capsa alpicola and Phormidium molle . Activity of the enzymes phosphoglycerate (PGA) dehydrogenase (EC 1.1.1.95), phosphoserine transaminase (EC 2.6.1.52) and phosphoserine phosphatase (EC 3.1.3.3) was detected in vitro in all species, but no PGA phosphatase (EC 3.1.3.20) or hydroxypyruvate reductase (EC 1.1.1.26) activity could be detected. Metabolism of [1-¹⁴C]PGA by cell-free extracts of C. peniocystis resulted in the labelling of serine, alanine and aspartate, which represented 84, 11 and 4% of the labelled amino acid pool, respectively, Labelled serine isolated from these experiments was 100% carboxyl-labelled indicating that it was formed directly from PGA. The labelling of serine was markedly reduced by 5 mM phosphoserine. These in vitro findings indicate that cyanobacteria are capable of synthesizing serine directly from PGA, independent of ribulose-1.5-bisphosphate oxidation, and that the route of serine synthesis via phosphohydroxypyruvate and phosphoserine is the predominant pathway in these organisms.     

Regulation of flavin biosynthesis in the methylotrophic yeast Hansenula polymorpha

Under various conditions of growth of the methylotrophic yeast Hansenula polymorpha, a tight correlation was observed between the levels of flavin adenine dinucleotide (FAD)-containing alcohol oxidase, and the levels of intracellularly bound FAD and flavin biosynthetic enzymes. Adaptation of the organism to changes in the physiological requirement for FAD was by adjustment of the levels of the enzymes catalyzing the last three steps in flavin biosynthesis, riboflavin synthetase, riboflavin kinase and flavin mononucleotide adenylyltransferase. The regulation of the synthesis of the latter enzymes in relation to that of alcohol oxidase synthesis was studied in experiments involving addition of glucose to cells of H. polymorpha growing on methanol in batch cultures or in carbon-limited continuous cultures. This resulted not only in selective inactivation of alcohol oxidase and release of FAD, as previously reported, but invariably also in repression/inactivation of the flavin biosynthetic enzymes. In further experiments involving addition of FAD to the same type of cultures it became clear that inactivation of the latter enzymes was not caused directly by glucose, but rather by free FAD that accumulated intracellularly. In these experiments no repression or inactivation of alcohol oxidase occurred and it is therefore concluded that the synthesis of this enzyme and the flavin biosynthetic enzymes is under separate control, the former by glucose (and possibly methanol) and the latter by intracellular levels of free FAD.Abbreviations FAD Flavin adenine dinucleotide - FMN riboflavin-5-phosphate; flavin mononucleotide - Rf riboflavin     

Enzymatic synthesis of high-molecular-mass poly-gamma-glutamate and regulation of its stereochemistry

For the first time, we succeeded in synthesizing in vitro poly-gamma-glutamate (PGA) with high molecular masses (>1,000 kDa) by the use of enzyme-associated cell membranes from Bacillus subtilis subsp. chungkookjang. The activity for PGA synthesis, however, was readily lost in the presence of critical concentrations of detergents tested in micelles. The optimum pH for the reaction was found to be approximately 7.0. We examined the effects of some divalent cations on PGA synthesis and found that Mg(2+) was essential in catalysis and that Zn(2+) additionally boosted the activity. In contrast, Fe(2+) and Ca(2+) acted as inhibitors. Mn(2+) did not apparently influence the in vitro formation of PGA. DL-Glutamate (D isomer content, 60 to 80%) apparently served as the best substrate; d-Glutamate was preferable to the L isomer as a substrate. When D- and L-glutamate were used for the reaction, the elongated chains of PGAs were composed of the D- and L-isomers, respectively. Our results suggest that the stereochemical properties of enzymatically synthesized PGAs substantially depend on the stereochemistry (DL ratio) of glutamate as the substrate. Furthermore, genetic analysis indicated that all the pgsB, -C, and -A gene products, which are responsible for PGA production by B. subtilis cells, were also indispensable for enzymatic PGA synthesis.     

The Effects of Organic Solvent on the Ribosyl Transfer Reaction by Thermostable Purine Nucleoside Phosphorylase and Pyrimidine Nucleoside Phosphorylase from Bacillus stearothermophilus JTS 859

The effects of organic solvents on the reaction rate and equilibrium of the ribosyl transfer reaction catalyzed by thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859 were examined at 60°C. The reaction rate in the presence of 10% acetone was 1.6 times higher than that of the control. Acetone was the best organic solvent among those tested for accelerating the reaction rate without denaturing the enzymes. On the other hand, the reaction rate in the presence of 5% ethyl acetate was 1.5 times higher than that of the control. However the enzymes were denatured completely after 1 h incubation. Consequently, the acceleration was not attributed to the stabilization of the enzymes. The equilibrium constants of the reaction were not influenced by the presence of acetone, methyl or ethyl alcohols.     

Use of neural networks in the mathematical modelling of the enzymic synthesis of amoxicillin catalysed by penicillin G acylase immobilized in chitosan

This study focuses in the mathematical modelling of the enzymic synthesis of amoxicillin by the reaction of p-hydroxyphenylglycine methyl ester and 6-aminopenicillanic acid (6APA), catalyzed by penicillin G acylase (PGA) immobilized on glutaraldehyde-chitosan, at 25°C and pH 6.5. Previous work on the kinetics and mechanism of reaction showed that the use of neural networks seems to be an interesting alternative to simulate experimental data of antibiotic production. Therefore, two feedforward neural networks, with one hidden layer, were trained and used to forecast the rates of amoxicillin and p-hydroxyphenylglycine (POHPG) net production. First of all, some parameters that affect the network performed were investigated, such as the number of nodes between the input and hidden layers and the number of interactions during the learning phase. Afterwards, hybrid models that coupled artificial neural networks to mass-balance equations were used to reproduce the performance of batch reactors for the production of amoxicillin. This approach provided accurate results, within the range of substrate concentration studied.