Protein engineering of alcohol dehydrogenase--1. Effects of two amino acid changes in the active site of yeast ADH-1

One of the promises held out by protein engineering is the ability to alter predictably the properties of an enzyme to enable it to find new substrates or catalyse existing substrates more efficiently, such manipulations being of interest both enzymologically and, potentially, industrially. It has been postulated that in yeast alcohol dehydrogenase (YADH-1) certain amino acids such as Trp 93 and Thr 48 constrict the active site due to their bulky side chains and thus impede catalysis of molecules larger than ethanol. To study effects of enlarging the active site we have made two changes into YADH-1, replacing Trp 93 with Phe and Thr 48 with Ser. Kinetic experiments showed that this enzyme had marked increases in reaction velocity for the n-alcohols propanol, butanol, pentanol, hexanol, heptanol, octanol and cinnamyl alcohol compared to the parent, agreeing with the prediction that expanding the active site should facilitate the oxidation of larger alcohols. The substrate affinities were slightly reduced in the altered enzyme, possibly due to its having reduced hydrophobicity at Phe 93.     

Short-chain alkanols and the functional efficiency of the Ca pump in the sarcoplasmic reticulum of rabbit skeletal muscles

The effect of 10 low molecular mass alkanols on the activity of Ca-ATPase (EC 3.6.1.38), Ca uptake and Ca efflux as well as the functional efficiency of the Ca pump in the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied. Some alkanols, especially when taken at low concentration, have been found to stimulate the activity of the Ca pump and Ca-ATPase, namely tert-butanol, isopropanol and ethanol (from the group of hydrophilic alkanols), and pentanol, isopentanol and hexanol (from the more hydrophobic alkanols). Methanol (from the first group) and isobutanol, butanol and propanol (from the second) do not stimulate the Ca pump compared with the control. The specific effect of different alkanols cannot be explained in terms of a unitary mechanism based on 'fluidity' changes of the membrane. It is assumed that, at low concentrations, certain alkanols (or groups of related alkanols) are able to promote the specific transition of membrane proteins into the active state, whereas at higher concentrations all alkanols provide for the non-functional state of the proteins.     

Kinetic characterization of yeast alcohol dehydrogenases. Amino acid residue 294 and substrate specificity

A three-dimensional model of yeast alcohol dehydrogenase, based on the homologous horse liver enzyme, was used to compare the substrate binding pockets of the three isozymes (I, II, and III) from Saccharomyces cerevisiae and the enzyme from Schizosaccharomyces pombe. Isozyme I and the S. pombe enzyme have methionine at position 294 (numbered as in the liver enzyme, corresponding to 270 in yeast), whereas isozymes II and III have leucine. Otherwise the active sites of the S. cerevisiae enzymes are the same. All four wild-type enzymes were produced from the cloned genes. In addition, oligonucleotide-directed mutagenesis was used to change Met-294 in alcohol dehydrogenase I to leucine. The mechanisms for all five enzymes were predominantly ordered with ethanol (but partially random with butanol) at pH 7.3 and 30 degrees C. The wild-type alcohol dehydrogenases and the leucine mutant had similar kinetic constants, except that isozyme II had 10-20-fold smaller Michaelis and inhibition constants for ethanol. Thus, residue 294 is not responsible for this difference. Apparently, substitutions outside of the substrate binding pocket indirectly affect the interactions of the alcohol dehydrogenases with ethanol. Nevertheless, the substitution of methionine with leucine in the substrate binding site of alcohol dehydrogenase I produced a 7-10-fold increase in reactivity (V/Km) with butanol, pentanol, and hexanol. The higher activity is due to tighter binding of the longer chain alcohols and to more rapid hydrogen transfer.     

Biosynthese des produits volatils de la pomme: Formation des alcools et des esters a partir des acides gras

The biosynthesis of apple volatiles (alcohols and esters) was studied using disks of aged tissues. After adding each substrate, the volatiles formed were analysed and estimated by GLC and their qualitative and quantitative composition compared with those of the control. Alcohols were formed from aliphatic acids having either the same number of carbon atoms, or from higher homologues through β-oxidation. Fatty acids with an even carbon number gave rise to butanol and hexanol, while odd carbon fatty acids generated propanol and pentanol. Esters were synthesized from the corresponding acid and alcohol and the yield was very high. Volatile profiles differed from one variety to another, yellow-skinned varieties producing chiefly acetic acid esters and the red-skinned mainly butyric acid esters. Provided the right substrates were given, all the esters were synthesized by the different tissues assayed, and the nature of the volatiles produced by each variety depends on the substrates present in the fruit. However, in Golden Delicious apples, which have a low content of butyric esters, erogenous butyrate was rapidly and completely transformed into acetate.     

直链脂肪醇对豚鼠乳头肌慢动作电位的影响

本工作用相平面法观察和分析了乙醇、丁醇、己醇和辛醇对豚鼠乳头肌慢动作电位(APA,APD50,APD90,(?)_(max),(?)′_(max))及其所对应的钙电流(I_(max))和钾电流(I′_(max))的影响。结果表明:这四种脂肪醇对钙电流均有抑制作用,而对钾电流的影响,随碳原子数的加大从增强(乙醇)逐渐转化为削弱(已醇,辛醇)。并且碳链越长,产生这些效应所需脂肪醇的浓度越低,这提示醇的作用可能与它们的疏水性相关。     

The actions of intermediate and long-chain n-alkanols on unitary NMDA currents in hippocampal neurons.

The actions of the n-alkanols butanol, pentanol, and octanol on unitary currents passing through N-methyl-D-aspartate (NMDA) ion channels have been studied in cultured CA1 hippocampal neurons. The cell-attached patch clamp method, with L-homocysteic acid included in the patch pipette, was used to record single channel NMDA currents at the cell resting potential or for hyperpolarizing patch potentials. With the n-alkanols added to the bath solution, the mean open times for the NMDA channel were diminished and the channel conductance was unchanged. A decrease in mean open time to about 70% of control value was found with butanol (3 mM), pentanol (1 mM), and octanol (0.02 mM). In addition the n-alkanols had small effects to decrease the frequency of channel openings and to increase the amplitude of the unitary currents. The effects of the alcohols on intracellular calcium levels, during NMDA applications, were also measured using the fluorescent dye FURA II.     

Effects of anesthetic alcohols on membrane transport processes in human erythrocytes

1. 1. Anesthetic alcohols (pentanol, hexanol and heptanol) were found to increase the fluidity of red cell membrane lipids as monitored by the fluorescence depolarization of diphenylhexatriene. The relative potency of the alcohols was found to be parallel to their relative membrane/water partition coefficients.

2. 2. Hexanol had biphasic effect on erythritol uptake by simple diffusion by red cells. At concentrations less than 9 mM, hexanol had no significant effect. At concentrations greater than 9 mM, there was an approximately linear increase in erythritol permeability with increasing alcohol concentration.

3. 3. The facilitated transport of uridine was markedly inhibited by hexanol. Hexanol at 6 mM produced a 65% inhibition of uridine (4 mM) uptake. Hexanol decreased both the apparent K_m and V values for the equilibrium exchange of uridine.

4. 4. The facilitated transport of galactose was only slightly inhibited by hexanol.

5. 5. Hexanol was without effect on the passive and active fluxes of Na⁺ and K⁺ in red cells with altered cation contents. Cells that were slightly depleted of K⁺ and cells that were highly K⁺-depleted were both insensitive to hexanol.

The effect of alcohols on guanosine 5'-diphosphate-3'-diphosphate metabolism in stringent and relaxed Escherichia coli

The effects of a series of alcohols on the stringent response system of Escherichia coli were studied. The alcohols used could be divided into two groups on the basis of the response of pppGpp and ppGpp to the growth downshift induced by the alcohols. The cells responded to the alcohols, methanol, ethanol, and propanol, as if they were being starved of amino acids. In the stringent strain CP78 these alcohols induced pppGpp and ppGpp accumulation and curtailed RNA synthesis, whereas in the relaxed strain CP79, both of these responses were absent. It was determined that this response was most likely due to an interference by these alcohols with the uptake of amino acids required by these strains. By contrast both stringent and relaxed cells elevated their level of ppGpp and decreased RNA accumulation when treated with butanol or pentanol. This response is similar to the effect of carbon source limitation. It was determined that the elevation of ppGpp in the stringent strain was primarily the result of increased ppGpp synthesis in response to these alcohols. In the relaxed strain the rise in ppGpp was dependent on a decrease in ppGpp degradation coupled with a moderate increase in ppGpp synthesis. This stimulation of ppGpp synthesis in relaxed cells, although small, suggests the existence of an enzyme distinct from stringent factor which is capable of synthesizing ppGpp. Data are presented which suggest that the activity of this enzyme is coupled to the potential for protein synthesis and energy availability of the cell, perhaps being regulated by the overall ratio of unchanged to amino-acylated tRNA.     

Molecular simulation of the effects of alcohols on peptide structure

The effects of alcohols on local protein structure have been simulated using computational approaches and model peptides. Molecular simulations were carried out on a 7-residue peptide created in both an extended conformation and an alpha-helix to explore alcohol-induced changes in peptide structure. It was assumed that alcohols hydrogen bond at peptide carbonyl groups with an optimum geometry and compete with water molecules at these site. Energy minimization of the peptide/alcohol assemblies revealed that alcohols induced a twist in the peptide backbone as a function of (1) the methylene chain length, (2) the hydrogen-bond geometry, (3) halogenation of the molecule, (4) concentration, and (5) the dielectric constant. The rank ordering of the potencies of the alcohols was hexafluoroisopropanol > trifluoroethanol approximately pentanol > butanol > ethanol > methanol. Helix destabilization by cosolvent was measured by examining the hydrogen-bond lengths in peptide structures that resulted from a combination of energy minimization and molecular dynamics simulations. Destabilization was also found to be dependent upon the chemical nature of the alcohol and the hydrogen-bond geometry. The data suggest that alcohols at low concentrations affect protein structure mainly through a combination of hydrogen-bonding and hydrophobic interactions that are influenced by the properties of the solvent.     

Alcohol adsorption on softwood lignin from aqueous solutions

Lignin prepared by acid and enzyme hydrolysis of a softwood mixture adsorbs acetone, butanol, and other alcohols while showing only a slight uptake of glucose. Adsorption of butanol is independent of temperature in the range of 30-65 degrees C. The Polanyi theory fits adsorption for the linear alcohols methanol through hexanol with values of AS and Delta(mu) ranging from 2.6 to 26 J mol(-1) K(-1)and -0.8 to -8 kJ/mol. The adsorption capacity is given by Q (g alcohol/g lignin) = KC(*). Where C(*) is the equilibrium alcohol concentration (g/mL), K = epsilon(W)exp (Delta/R), and epsilon(w) is the porosity of the lignin (0.23-0.42 mL/g). The value of the adsorption capacity constant K for n-butanol ranges from 1.3 to 2.7 mL/g on sorbent containing 26-72% lignin, while ethanol is 0.5-0.73, acetone is 0.62-1.0, and glucose is 0.35. Adsorption is shown to occur through combined hydrophobic and hydrophilic interactions of the alkyl and hydroxyl groups, respectively, of the adsorbate with the lignin. Consequently, for the alcohols methanol to hexanol, we present the capacity constant K[=K(R) + K(OH)] as a sum of an alky! adsorption constant (0.1-9.5 mL/g) and a hydrophilic (0.40-0.50 mL/g) contribution. This approach may be applicable to organic acids. Lignin's sorbent properties have potential to moderate product inhibition in the anaerobic acetone-butanol-ethanol (ABE) fermentation.     

Enantioselective synthesis of ibuprofen esters in AOT/isooctane microemulsions by Candida cylindracea lipase

The enantioselective esterification of racemic ibuprofen, catalyzed by a Candida cylindracea lipase, was studied in a water-in-oil microemulsion (AOT/isooctane). By using n-propanol as the alcohol, an optimal W(0) ([H(2)O]/[AOT] ratio) of 12 was found for the synthesis of n-propyl-ibuprofenate at room temperature. The lipase showed high preference for the S(+)-enantiomer of ibuprofen, which was esterified to the corresponding S(+)-ibuprofen ester. The R(-)-ibuprofen remained unesterified in the microemulsion. The calculated enantioselectivity value (E) for S-ibuprofen ester was greater than 150 (conversion 0.32). The enzyme activities of n-alcohols with different chain lengths (3-12) were compared, and it appeared that short- (propanol and butanol) and long-chained (decanol and dodecanol) alcohols were better substrates than the intermediate ones (pentanol, hexanol, and octanol). However, unlike secondary and tertiary alcohols, all of the tested primary alcohols were substrates for the lipase. The reversible reaction (i.e., the hydrolysis of racemic ibuprofen ester in the microemulsion) was also carried out enantioselectively by the enzyme. Only the S form of the ester was hydrolyzed to the corresponding S-ibuprofen. The reaction yield was, however, only about 4% after 10 days of reaction. The corresponding yield for the esterification of ibuprofen was about 35% (10 days). The high enantioselectivity displayed by the lipase in the microemulsion system was seen neither in a similar esterification reaction in a pure organic solvent system (isooctane) nor in the hydrolysis reaction in an aqueous system (buffer). The E value for S-ibuprofen ester in the isooctane system was 3.0 (conversion 0.41), and only 1.3 for S-ibuprofen in the hydrolysis reaction (conversion 0.32). The differences in enantioselectivity for the lipase in various systems are likely due to interfacial phenomena. In the microemulsion system, the water in which the enzyme is dissolved is separated from the solvent by a layer of surfactant molecules, thus creating an interface with a relatively large area. Such interfaces are not present in the pure organic solvent systems (no surfactant) nor in aqueous systems. (c) 1993 John Wiley & Sons, Inc.     

Ethanol inhibits mitogen-induced calcium mobilization in mouse splenocytes.

Ethanol inhibited the mitogen-induced initial increase in cytoplasmic free-calcium [Ca2+]i in mouse splenocytes. This effect was concentration-dependent, reversible, and observed at pharmacologically relevant concentrations (24-166mM). Other short-chain alcohols such as propanol, butanol, and pentanol also inhibited this mitogen-induced increase in [Ca2+]i. The potencies of these alcohols to produce this effect were highly correlated (r = 0.98, p less than 0.001) with their membrane/buffer partition coefficients. Analysis of mouse splenocyte subpopulations demonstrated that this effect was manifest in both B and T lymphocytes. Within T lymphocyte subpopulations, both CD4+ and CD8+ T cells were affected. These results suggest that the inhibition of [Ca2+]i increase may be an early event mediating ethanol-induced immunosuppression and that this may be a predisposing factor to infection and malignancies associated with alcoholism.     

Production of biofuels from C1-gases with Clostridium and related bacteria—Recent advances

Clostridium spp. are suitable for the bioconversion of C₁-gases (e.g., CO₂, CO and syngas) into different bioproducts. These products can be used as biofuels and are reviewed here, focusing on ethanol, butanol and hexanol, mainly. The production of higher alcohols (e.g., butanol and hexanol) has hardly been reviewed. Parameters affecting the optimization of the bioconversion process and bioreactor performance are addressed as well as the pathways involved in these bioconversions. New aspects, such as mixotrophy and sugar versus gas fermentation, are also reviewed. In addition, Clostridia can also produce higher alcohols from the integration of the Wood-Ljungdahl pathway and the reverse ß-oxidation pathway, which has also not yet been comprehensively reviewed. In the latter process, the acetogen uses the reducing power of CO/syngas to reduce C₄ or C₆ fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol.     

Differential effects of ethanol and hexanol on the Escherichia coli cell envelope.

Both ethanol and hexanol inhibited the growth of Escherichia coli, but their effects on the organization and composition of the cell envelope were quite different. Hexanol (7.8 x 10(-3) mM) increased membrane fluidity, whereas ethanol (0.67 M) had little effect. During growth in the presence of ethanol, the proportion of unsaturated fatty acids increased. The opposite change was induced by hexanol. Unlike hexanol, growth in the presence of ethanol resulted in the production of un-cross-linked peptidoglycan with subsequent lysis. Salt (0.3 M) protected cells against ethanol-induced lysis but potentiated growth inhibition by hexanol. Mutants isolated for resistance to ethanol-induced lysis synthesized cross-linked peptidoglycan during growth in the presence of ethanol but remained sensitive to hexanol. A general hypothesis was presented to explain the differential effects of ethanol and hexanol. All alcohols are viewed as similar in having both an apolar chain capable of interacting with hydrophobic environments and a hydroxyl function capable of hydrogen bonding. The differential effects of short-chain alcohols may represent effects due to the high molar concentrations of hydrogen bonding groups with an apolar end within the environment. These may replace bound water in some cases. With longer-chain alcohols such as hexanol, the effects of the acyl chain would dominate, and limitations of solubility and cellular integrity would mask these hydroxyl effects.     

Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry

Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO₂-gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.     

Alcohol-induced biphasic inhibition of myosin subfragment 1 K-EDTA-ATPase

Butanol-induced inhibition of K-EDTA-ATPase of myosin subfragment 1 proceeded by biphasic kinetics, consisting of rapid and slow inactivations. The extent of the rapid inactivation, which was estimated by extrapolating the process of slow inactivation to zero time of the incubation period, was saturated with butanol concentration. Recovery of activity by dilution in the rapid phase indicates that the rapid process is reversible. The slow inactivation was concomitant with a partial denaturation of the 50 kDa domain of S1, which was detected by limited tryptic digestion. Other alcohols (methanol, ethanol, propanol and hexanol) also inhibited the K-EDTA-ATPase in the rapid phase. The Ki decreased with an increase in the number of methylene groups of alcohol. When K-EDTA-ATPase activity in the rapid phase was plotted against viscosity, surface tension or dielectric constant, the curves were different for each of the various alcohol solutions. The rapid inactivation appears to be caused by a binding of the alkyl group to S1, rather than by solvent effects. The kinetics of rapid butanol inhibitions indicate that butanol reduces the maximum activity of ATPase but enhances an apparent affinity of S1 with ATP. These indications suggest that alcohol stabilizes S1.KATP intermediate. The rapid K-EDTA-ATPase inhibition was observed at the same alcohol concentration where S1 Mg-ATPase was activated.     

Effects of chronic administration of either ethanol or pentanol on rat duodenum morphology

The morphology of the rat duodenum after chronic treatment with 15% (v/v) ethanol and 4% (v/v) pentanol was studied. Male Wistar rats of experimental groups were given ethanol and pentanol for 15 weeks with food and fluid freely available. Ethanol-15% and 4% pentanol-fed rats showed a significantly reduced fluid and food intake as compared with control rats. The study of the mucosa indicated that the number of chronic inflammatory infiltrating (mononuclear cells) and goblet cells was higher in the groups of the ethanol- and pentanol-fed rats than in the control group. There was an increase in the thickness of the brush border in pentanol-fed rats. Intervillus adhesion was concurrently observed in the pentanol-fed rats but not in the control or ethanol-fed rats. After ethanol feeding many of the villi developed blebs at the apex of the villus or laterally on its upper half. These blebs generally remained intact. In contrast, after pentanol feeding no bleb formation was appreciated. The intake of ethanol and other short chain alcohols present in alcoholic beverages leads to mainfold disturbances on the rat duodenum. These findings suggest that the chronic ingestion of pentanol seems to promote cellular changes but less important than those observed after chronic ethanol ingestion.     

An alcohol acyl transferase from apple (cv. Royal Gala), MpAAT1, produces esters involved in apple fruit flavor

Apple flavor is characterized by combinations of ester compounds, which increase markedly during fruit ripening. The final step in ester biosynthesis is catalyzed by alcohol acyl transferases (AATs) that use coenzyme A (CoA) donors together with alcohol acceptors as substrates. The gene MpAAT1, which produces a predicted protein containing features of other plant acyl transferases, was isolated from Malus pumila (cv. Royal Gala). The MpAAT1 gene is expressed in leaves, flowers and fruit of apple. The recombinant enzyme can utilize a range of alcohol substrates from short to medium straight chain (C3-C10), branched chain, aromatic and terpene alcohols. The enzyme can also utilize a range of short to medium chain CoAs. The binding of the alcohol substrate is rate limiting compared with the binding of the CoA substrate. Among different alcohol substrates there is more variation in turnover compared with K(m) values. MpAAT1 is capable of producing many esters found in Royal Gala fruit, including hexyl esters, butyl acetate and 2-methylbutyl acetate. Of these, MpAAT1 prefers to produce the hexyl esters of C3, C6 and C8 CoAs. For the acetate esters, however, MpAAT1 preference depends upon substrate concentration. At low concentrations of alcohol substrate the enzyme prefers utilizing the 2-methylbutanol over hexanol and butanol, while at high concentrations of substrate hexanol can be used at a greater rate than 2-methylbutanol and butanol. Such kinetic characteristics of AATs may therefore be another important factor in understanding how the distinct flavor profiles of different fruit are produced during ripening.     

Membrane fluidization by alcohols inhibits DesK-DesR signalling in Bacillus subtilis

After cold shock, the Bacillus subtilis desaturase Des introduces double bonds into the fatty acids of existing membrane phospholipids. The synthesis of Des is regulated exclusively by the two-component system DesK/DesR; DesK serves as a sensor of the state of the membrane and triggers Des synthesis after a decrease in membrane fluidity. The aim of our work is to investigate the biophysical changes in the membrane that are able to affect the DesK signalling state. Using linear alcohols (ethanol, propanol, butanol, hexanol, octanol) and benzyl alcohol, we were able to suppress Des synthesis after a temperature downshift. The changes in the biophysical properties of the membrane caused by alcohol addition were followed using membrane fluorescent probes and differential scanning calorimetry.We found that the membrane fluidization induced by alcohols was reflected in an increased hydration at the lipid-water interface. This is associated with a decrease in DesK activity. The addition of alcohol mimics a temperature increase, which can be measured isothermically by fluorescence anisotropy. The effect of alcohols on the membrane periphery is in line with the concept of the mechanism by which two hydrophilic motifs located at opposite ends of the transmembrane region of DesK, which work as a molecular caliper, sense temperature-dependent variations in membrane properties.     

Norcantharimide analogues possessing terminal phosphate esters and their anti-cancer activity

A family of norcantharidin analogues possessing a terminal alcohol (ethanol, propanol, butanol, pentanol, hexanol and cyclohexanol) moiety were treated with either chlorodiethyl, chlorodiphenyl or chloro-bis-trichloroethyl-phosphate to afford highly focused libraries of the corresponding phosphate esters. Subsequent biological screening against a panel of nine human cancer cell lines identified a trend between the ease of phosphate unmasking (phosphate ester hydrolysis) and cell death. The most potent analogues possessed either a diphenyl or a bis-trichloroethyl moiety. The effect of alkyl spacer was also examined with the hexyl analogues typically more potent. 4-Aza-4-(3-{bis(2,2,2-trichloroethyl)phosphate}propyl)-10-oxatricyclo[5.2.1.0]decane-3,5-dione (10b) was the most potent analogue synthesised with an average GI(50) of 11 μM across a panel of nine human carcinoma cell lines: colon carcinoma (HT29 and SW480); breast carcinoma (MCF-7); ovarian carcinoma (A2780); lung carcinoma (H460); skin carcinoma (A431); prostate carcinoma (DU145); neuronal carcinoma (BE2-C) and brain carcinoma (SJ-G2). This represents a fivefold improvement in anti-proliferative activity relative to the lead, norcantharidin.