Studies on lysophospholipases: VII. Synthesis of acylthioester analogs of lysolecithin and their use in a continuous spectrophotometric assay for lysophospholipases,a method with potential applicability to other lipolytic enzymes

The synthesis of acylthioester analogs of lysolecithins, i.e., 2-hexadecanoylthio-1-ethyl-phosphorylcholine and 3-hexadecanoylthio-1-propyl-phosphorylcholine is described. Both compounds were found to be hydrolysed by a homogeneous lysophospholipase from beef liver, a spectrophotometric assay for the activity of which was developed by continuous measurement of the released thiol groups in the presence of dithionitrobenzoic acid.Phospholipase A₂ from pig pancreas effected hydrolysis of the acylthioester bond in 2-hexadecanoylthio-1-ethyl-phosphorylcholine, the enzymatic action of which could also be monitored spectrophotometrically.Lipase from pig pancreas was found to hydrolyse acylthioester bonds in 2-hexadecanoylthio-1-ethanol. The tributyryl ester of 3-mercapto-1,2-propanediol was synthesized and used to compare the release of total acid and thiol groups during hydrolysis with lipase. A ratio of about 2:1 was found for these releases.These findings clearly indicate the potential applicability of acylthioester analogs of substrates for phospholipases, lysophospholipases, and lipases in continuous spectrophotometric assays for lipolytic enzymes.     

Synthesis of choline and ethanolamine phospholipids with thiophosphoester bonds as substrates for phospholipase C.

Spectrophotometric assays of esterases are sensitive, rapid, and quite specific when thioester substrates are used. Glycerophospholipids with thiophosphoester bonds may be useful as substrates for phospholipase C (EC 3.1.4.3). These have been made from mercaptoglycerol and mercaptoethanol. The thiols were oxidized to disulfides, acylated, and reduced with dithiothreitol. Phosphocholine derivatives were made by the classical methods for oxyphosphoesters. The phosphatidyl choline analogue was converted to the phosphatidyl ethanolamine analogue by transphosphatidylation with cabbage phospholipase D and ethanolamine. Structures were proved with enzymic hydrolysis, infrared spectra, TLC behavior, and elemental analyses. The synthesized compounds were rac-1-S-phosphocholine-2,3-O-didecanoyl-1-mercapto-2,3-propanediol, 1-S-phosphoethanolamine-2,3-O-didecanoyl-1-mercapto-2,3-propanediol, and 1-S-phosphocholine-2-O-hexadecanoyl-1-mercapto-2-ethanol.     

Substrate specificity of coenzyme B12-dependent diol dehydrase: glycerol as both a good substrate and a potent inactivator.

The substrate specificity of adenosylcobalamin-dependent diol dehydrase was further studied in detail using an enzyme preparation that appears homogeneous by ultracentrifugal and gel electrophoretical criteria. Besides 1,2-propanediol and 1,2-ethanediol, glycerol, 1,2- and 2,3-butanediol were found to serve as substrate for the enzyme, whereas 1,3-propanediol was not. Of the substrate analogs tested, glycerol displayed some striking features: it was dehydrated to β-hydroxypropionaldehyde with concomitant inactivation of the enzyme. Although the initial velocity with glycerol was comparable to that with 1,2-propanediol, the dehydration reaction ceased almost completely within 3 min accompanying rapid, irreversible inactivation of the holoenzyme. 1,2- and 2,3-Butanediol were converted to butyraldehyde and methyl ethyl ketone, respectively, at a rate much lower than that with 1,2-propanediol. 2,3-Butanediol is the only compound, other than 1,2-diols, known at present to show a considerable substrate activity.     

Loss of stereospecificity of phospholipases C and D upon introduction of a 2-alkyl group into rac-1,2-diacylglycero-3-phosphocholine

rac-1-[1-14C]Lauroyl-2-oleylglycero-3-phospho[methyl-3H]choline and rac-1-lauroyl-2-[1-14C]oleoylglycero-3-phospho[methyl-3H]choline along with rac-1-palmitoyl-2-oleylglycero-3-phosphocholine and sn-1-palmitoyl-2-oleylglycero-3-phosphocholine were synthesized and subjected to hydrolysis with phospholipase C (EC 3.1.4.3) from Clostridium perfringens and phospholipase D (EC 3.1.4.4) from cabbage. Kinetics of hydrolysis of the radioactive substrates were determined by measuring the 3H radioactivity retained in the aqueous phase due to free choline and phosphocholine and the 3H and 14C radioactivity recovered in the organic phase due to the released diacylglycerols and phosphatidic acids and the residual phosphatidylcholines. The rate of hydrolysis of the unlabelled substrates by phospholipase C was determined by thin-layer chromatography and gas-liquid chromatography of the methanolysis products. The relative initial rates of hydrolysis of sn-1,2,- and sn-2,3-enantiomers were 100-200:1 for phospholipase C and 40-50:1 for phospholipase D using rac-1-lauroyl-2-oleoylglycero-3-phosphocholine as the substrate. The substitution of the 2-acyl group by an alkyl group resulted in a loss of stereospecificity, which was partial for phospholipase C (relative rates equal to 8-13:1) and total for phospholipase D. There was a parallel dramatic decrease (500-1000-fold) in the initial rate of hydrolysis with phospholipase C but the activity of phospholipase D was only moderately reduced (18-fold). These findings are consistent with the earlier observed loss of the stereospecificity of lipoprotein lipase following introduction of a 2-alkyl group into triacylycerols, and point to a general unsuitability of 2-alkyl-linked acylglycerols as substrates for the assay of the stereospecificity of lipases, as well as for the isolation of enantiomeric 2-alkylacylglycerols by means of stereospecific lipases.     

Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2.

An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-1-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50 degrees C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.     

Haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26: X-ray crystallographic studies of dehalogenation of brominated substrates

The haloalkane dehalogenases are detoxifying enzymes that convert a broad range of halogenated substrates to the corresponding alcohols. Complete crystal structures of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), and complexes of LinB with 1,2-propanediol/1-bromopropane-2-ol and 2-bromo-2-propene-1-ol, products of debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, were determined from 1.8 A resolution X-ray diffraction data. Published structures of native LinB and its complex with 1,3-propanediol [Marek et al. (2000) Biochemistry 39, 14082-14086] were reexamined. The full and partial debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, conformed to the observed general trend that the sp(3)-hybridized carbon is the predominant electrophilic site for the S(N)2 bimolecular nucleophilic substitution in dehalogenation reaction. The 2-bromo-2-propene-1-ol product of 2,3-dibromopropene dehalogenation in crystal was positively identified by the gas chromatography-mass spectroscopy (GC-MS) technique. The 1,2-propanediol and 1-bromopropane-2-ol products of 1,2-dibromopropane dehalogenation in crystal were also supported by the GC-MS identification. Comparison of native LinB with its complexes showed high flexibility of residues 136-157, in particular, Asp146 and Glu147, from the cap domain helices alpha(4) and alpha(5)('). Those residues were shifted mainly in direction toward the ligand molecules in the complex structures. It seems the cap domain moves nearer to the core squeezing substrate into the active center closer to the catalytic triad. This also leads to slight contraction of the whole complex structures. The flexibility detected by crystallographic analysis is in remarkable agreement with flexibility observed by molecular dynamic simulations.     

Opioid Peptides from Human β-Casein

A bacterium that assimilates 2,3-dichloro-1-propanol was isolated from soil by enrichment culture. The strain was identified as Pseudomonas sp. by the taxonomic studies. The strain converted 2,3-dichloro-1-propanol to 3-chloro-1,2-propanediol, releasing chloride ion. The conversion was stereospecific because the residual 2,3-dichloro-1-propanol and formed 3-chloro-1,2-propanediol gave optical rotation. The resting cells converted various halohydrins to the dehalogenated alcohols, and cell-free extracts had strong epoxyhydrolase activity. These results indicated that the strain assimilated 2,3-dichloro-1-propanol via 3-chloro-1,2-propanediol, glycidol, and glycerol. The possibility to manufacture optically active 2,3-dichloro-1-propanol is discussed.     

Cryoprotection of red blood cells by a 2,3-butanediol containing mainly the levo and dextro isomers.

A 2,3-butanediol containing 96.7% (w/w) racemic mixture of the levo and dextro isomers and only 3.1% (w/w) of the meso isomer (called 2,3-butanediol 97% dl) has been used for the cryoprotection of red blood cells. The erythrocytes were cooled to -196 degrees C at rates between 2 and 3500 degrees C/min, followed by slow or rapid warming. Up to 20% (w/w) of this polyalcohol, only the classical peak of survival is observed, as with up to 20% (w/w) 1,2-propanediol or 1,3-butanediol. Twenty percent 2,3-butanediol 97% dl can protect red blood cells very efficiently. The maximum survival, of 90%, as with 20% glycerol, is a little lower than with 20% 1,2-propanediol and higher than with 20% 1,3-butanediol. Fifteen percent 2,3-butanediol protects fewer red blood cells than 15% glycerol or 1,2-propanediol, with a maximum survival of about 80%. The best cryoprotection by 30% 2,3-butanediol 97% dl is obtained at the slowest cooling and warming rates, where survival approaches 90%. After a minimum, an increase of survival is observed at the fastest cooling rates, which would correspond to complete vitrification. These rates are lower than with 30%, 1,2-propanediol or 1,3-butanediol, in agreement with the higher glass-forming tendency of 2,3-butanediol 97% dl solutions. In agreement with the remarkable physical properties of its aqueous solutions, the present experiments also suggest that 2,3-butanediol containing mainly the levo and dextro isomers could be a very useful cryoprotectant for organ cryopreservation. However, it would perhaps be better to use it in combination with other cryoprotectants, since it is a little more toxic than glycerol or 1,2-propanediol at high concentrations.     

Identification and characterization of a mycobacterial (2R,3R)-2,3-butanediol dehydrogenase

Bacterial strain B-009, capable of using racemic 1,2-propanediol (PD), was identified as a rapid-growing member of the genus Mycobacterium. The strain is phylogenetically related to M. gilvum, but has slightly different physiological characteristics. An NAD(+)-dependent enantioselective alcohol dehydrogenase, which acts on R-PD, was purified from the strain. The enzyme was a homodimer of a peptide coded by a 1047-bp gene (mbd1). A highly conserved sequence for medium-chain dehydrogenase/reductases with a preference for secondary alcohols was found in the gene. Hydroxyacetone was produced from R-PD by an enzymatic reaction, indicating that position 2 of the substrate was oxidized. The enzyme activity was highest for (2R,3R)-2,3-butanediol (R,R-BD), enabling the enzyme to be identified as (2R,3R)-2,3-butanediol dehydrogenase (R,R-BD-DH). A homology search revealed M. gilvum, M. vanbaalenii, and M. semegmatis to have ORFs similar to mbd1, suggesting the widespread distribution of genes encoding R,R-BD-DH among mycobacterial strains.     

Rhamnose-induced propanediol oxidoreductase in Escherichia coli: purification, properties, and comparison with the fucose-induced enzyme.

Escherichia coli are capable of growing anaerobically on L-rhamnose as a sole source of carbon and energy and without any exogenous hydrogen acceptor. When grown under such condition, synthesis of a nicotinamide adenine dinucleotide-linked L-lactaldehydepropanediol oxidoreductase is induced. The functioning of this enzyme results in the regeneration of nicotinamide adenine dinucleotide. The enzyme was purified to electrophoretic homogeneity. It has a molecular weight of 76,000, with two subunits that are indistinguishable by electrophoretic mobility. The enzyme reduces L-lactaldehyde to L-1,2-propanediol with reduced nicotinamide adenine dinucleotide as a cofactor. The Km were 0.035 mM L-lactaldehyde and 1.25 mM L-1,2-propanediol, at pH 7.0 and 9.5, respectively. The enzyme acts only on the L-isomers. Strong substrate inhibition was observed with L-1,2-propanediol (above 25 mM) in the dehydrogenase reaction. The enzyme has a pH optimum of 6.5 for the reduction of L-lactaldehyde and of 9.5 for the dehydrogenation of L-1,2-propanediol. The enzyme is, according to the parameters presented in this report, indistinguishable from the propanediol oxidoreductase induced by anaerobic growth on fucose.     

Influence of size and polarity of residue 31 in porcine pancreatic phospholipase A2 on catalytic properties

Residue 31 of porcine pancreatic phospholipase A2 (PLA2) is located at the entrance to the active site. To study the role of residue 31 in PLA2, six mutant enzymes were produced by site-directed mutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Ser or Gly. Direct binding studies indicated a three to six times greater affinity of the Trp31 PLA2 for both monomeric and micellar substrate analogs, relative to the wild-type enzyme. The other five mutants possess an unchanged affinity for monomers of the product analog n-decylphosphocholine and for micelles of the diacyl substrate analog rac-1,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine. The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholine were decreased 9-20 times for these five mutants. Kinetic studies with monomeric substrates showed that the mutants have Vmax values which range between 15 and 70% relative to the wild-type enzyme. The Vmax values for micelles of the zwitterionic substrate 1,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3-50 times. The Km values for the monomeric substrate and the Km values for the micellar substrate were hardly affected in the case of five of the six mutants, but were considerably decreased when Trp was present at position 31. The results of these investigations point to a versatile role for the residue at position 31: involvement in the binding and orientating of monomeric substrate (analogs), involvement in the binding of the enzyme to micellar substrate analogs and possibly involvement in shielding the active site from excess water.     

Attenuation of sn-1,2-diacylglycerol second messengers by diacylglycerol kinase. Inhibition by diacylglycerol analogs in vitro and in human platelets

Diacylglycerol kinase is though to play a central role in the metabolism of diacylglycerol second messengers in agonist-stimulated cells. A series of diacylglycerol analogs were tested for their ability to act as substrates or inhibitors of diacylglycerol kinase with the goal of determining the substrate specificity of the enzyme, and of discovering inhibitors. Screening of these compounds was performed using a partially purified diacylglycerol kinase from pig brain. Modified assays for this enzyme using co-sonicated mixtures of diacylglycerol and anionic phospholipids were developed. This enzyme was found to be quite specific for sn-1,2-diacylglycerol (KM 24 microM for dioctanoyl-glycerol). Among the analogs investigated, only 1,2-dioctanoyl-2-amino-1,3-propanediol was utilized at a significant rate. Two analogs, dioctanoylethylene glycol (KI 58 microM) and 1-monooleoylglycerol (KI 91 microM), were potent inhibitors in vitro. These compounds were tested for effects on diacylglycerol formation and metabolism in thrombin-stimulated human platelets. Dioctanoylethylene glycol inhibited diacylglycerol phosphorylation in platelets (70-100% at 100 microM) leading to a longer-lived diacylglycerol signal. This compound may be a useful tool for studies of diacylglycerol kinase in other cell types. 1-Monooleoylglycerol treatment elevated diacylglycerol levels up to 4-fold in unstimulated platelets and up to 10-fold in thrombin-stimulated platelets. The implications with regard to the pathways of diacylglycerol metabolism in human platelets are discussed.     

Oxidation of 3-C-(2-amino-2-deoxy-D-glucopyranosyl)-1-propene compounds and the structure of 3-C-(2-amino-2-deoxy-D-glucopyranosyl)-1,2-propanediol derivatives for a synthesis of 2,3-didehydro-2,7-dideoxy-N-acetylneuraminic acid

Oxidation of 5-acetamido-4,8-anhydro-1,2,3,5-tetradeoxy-D-glycero-D-ido-non-1-enitol [3-C-(2-amino-2-deoxy-beta-D-glucopyranosyl)-1-propene] was studied to search for preparative routes to aminodeoxy didehydro nonulosonic acid derivatives. Since only moderate chiral induction was observed with osmium tetroxide dihydroxylation as well as with peracid epoxidation, the catalytic asymmetric dihydroxylation conditions were applied to give the stereocontrolled formation of 1,2-propanediol derivatives. The structures of these diastereoisomeric 1,2-propanediol derivatives were determined by X-ray crystallographic analyses. The formation of diastereoisomeric 1,2-propanediols also varied with the nature of 2-substituent on the aminodoexy glycosyl moiety. Thus 5-acetamido-4,8-anhydro-3,5-dideoxy-D-erythro-L-ido-nonitol [(2S)-3-C-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-1,2-propanediol] was obtained predominantly up to 70% from 3-C-(2-acetamido-2-deoxyglycosyl)-1-propene by the use of ADmixbeta reagent. The (2S)-propanediol derivative was transformed in a five-step reaction sequence to 2,3-didehydro-2,7-dideoxy-N-acetylneuraminic acid.     

Primary stimuli of icosanoid release inhibit arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. Mechanism of action of hydrogen peroxide and methyl mercury in platelets

Icosanoid formation in platelets depends on the concentration of free arachidonate that is mainly liberated from membrane phospholipids by phospholipase A2. The concentration of free arachidonate is also controlled by the activities of the reacylating enzymes arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. In human platelet microsomes we determined the high enzyme activities of 5.9 nmol.min-1.(10(9) platelets)-1 for the arachidonoyl-CoA synthetase and 37 nmol.min-1.(10(9) platelets)-1 for the lysophospholipid acyltransferase. The activities of these reacylating enzymes were strongly reduced by hydrogen peroxide (H2O2) and methyl mercury that are primary stimuli of arachidonate release in intact platelets. H2O2 inhibited the arachidonoyl-CoA synthetase with an IC50 of 3.3 mmol/l without affecting the lysophospholipid acyltransferase. Sulfhydryl group protection by 3-mercapto-1,2-propanediol did not overcome the inhibition but glutathione prevented the inhibition of the arachidonoyl-CoA synthetase by H2O2. This suggests that glutathione by virtue of the glutathione peroxidase reduces H2O2 rather than that it protects free sulfhydryl groups of the arachidonoyl-CoA synthetase. Methyl mercury left the arachidonoyl-CoA synthetase activity unaffected but inhibited the lysophospholipid acyltransferase activity with an IC50 of 3.4 mumol/l. The inhibition is probably evoked by the blockade of sulfhydryl groups of the lysophospholipid acyltransferase because it disappeared when 3-mercapto-1,2-propanediol was added at a concentration higher than that of methyl mercury. Thrombin as a physiological full agonist, Ca2+ less than or equal to 1 mmol/l, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol as model stimuli of protein kinase C neither influenced arachidonoyl-CoA synthetase nor lysophospholipid acyltransferase. It is concluded that the inhibitory effect of H2O2 and methyl mercury on the arachidonate-reacylating enzymes arachidonoyl-CoA synthetase or lysophospholipid acyltransferase, respectively, are responsible for their capacity to stimulate icosanoid release in intact cells. Thrombin and its intracellular messengers Ca2+ and diacylglycerol do not directly affect arachidonoyl-CoA synthetase and lysophospholipid acyltransferase.     

Ferrous-activated nicotinamide adenine dinucleotide-linked dehydrogenase from a mutant of Escherichia coli capable of growth on 1, 2-propanediol

A nicotinamide adenine dinucleotide-linked dehydrogenase has been partially purified from a mutant of Escherichia coli K-12 able to grow on l-1,2-propanediol as carbon and energy source. This enzyme catalyzes the dehydrogenation at carbon 1 of l-1,2-propanediol, glycerol, 1,3-propanediol, ethylene glycol, and ethyl alcohol. The purified protein requires added ferrous or managanous ions. The V(max) and the apparent K(m) for a given substrate vary with the particular metal used.     

Mechanism of hilA repression by 1,2-propanediol consists of two distinct pathways, one dependent on and the other independent of catabolic production of propionate, in Salmonella enterica serovar Typhimurium

A glycerol dehydrogenase gene was selected as a multicopy suppressor rescuing the reduced hilA expression in the Salmonella enterica serovar Typhimurium cpxA mutant. A substrate of the enzyme, 1,2-propanediol, repressed hilA expression. The 1,2-propanediol-mediated repression at 150 mM, but not that at 300 mM, was abrogated by blocking the catabolism producing propionate from 1,2-propanediol.     

Glass-forming tendency and stability of the amorphous state in the aqueous solutions of linear polyalcohols with four carbons. I. Binary systems water-polyalcohol

All the aqueous solutions of linear saturated polyalcohols with four carbons have been investigated at low temperature. Only ice has been observed in the solutions of 1,3-butanediol and 1,2,3- and 1,2,4-butanetriol. For same solute concentration, the glass-forming tendency on cooling is highest with 2,3-butanediol, where it is comparable to that with 1,2-propanediol, the best solute reported to date. However, the quantity of ice and hydrate crystallized is particularly high on slow cooling or on subsequent rewarming. The highest stability of the amorphous state is observed on rewarming the 1,2-butanediol and 1,3-butanediol solutions. With respect to this property, these compounds come just after 1,2-propanediol and before all the other compounds studied so far. They are followed by dimethylsulfoxide and 1,2,3-butanetriol. The glass-forming tendency of the 1,3-butanediol solutions is also very high; it is third only to that of 1,2-propanediol and 2,3-butanediol. The glass-forming tendency is a little smaller with 1,2-butanediol, but it is cubic instead of ordinary hexagonal ice which crystallizes on cooling rapidly with 35% 1,2-butanediol. Cubic ice is thought to be innocuous. A gigantic glass transition is observed with 45% of this strange solute. 1,4-Butanediol, 45% also favors cubic ice greatly. Therefore, 1,2- and 1,3-butanediol with comparable physical properties are perhaps as interesting as 1,2-propanediol for cryopreservation of cells or organs by complete vitrification. Together with 1,2-propanediol, 1,2- and 1,3-butanetriol, 1,2,3-butanetriol, and perhaps 2,3-butanediol provide an interesting battery of solutions for cryopreservation by vitrification.     

Inhibition of cytochrome P450 mediated enzyme activity by alkylphosphocholines

The inhibitory potency of four alkylphospholipids: rac-1-O-phosphocholine-2-hydroxy-octadecane (rac-2-OH), rac-1-O-phosphocholine-2-O-acetyl-octadecane (rac-2-O-acetyl), rac-1-O-phosphocholine-2-amino-octadecane (rac-2-NH2) and rac-1-O-phosphocholine-2-N-acetyloctadecane (rac-2-N-acetyl), on the cytochrome P450-dependent monooxygenase activity has been evaluated. The IC50 values of the alkylphosphocholines with 7-ethoxycoumarin as substrate in liver microsomal fractions of PB-treated rats and with a reconstituted CYP2B1: NADPH-P450-reductase system are in the range of 3.2-5.0 microM and 2.8-3.5 microM, respectively. Lineweaver-Burk plots with the inhibitors in concentrations that were found to cause roughly a 50% inhibition and with 7-ethoxycoumarin as substrate revealed for all four alkylphospholipids a competitive inhibition type. The degree of the competitive inhibition is quantified by the Ki values. With liver microsomal fractions of PB-treated rats, the Ki values of rac-2-OH (Ki = 1.36 microM) and rac-2-O-acetyl (Ki = 1.33 microM) differs slightly from those of rac-2-NH2 (Ki = 2.2 microM) and rac-2-N-acetyl (Ki = 2.2 microM), but with the reconstituted CYP2B1: NADPH-P450-reductase system all Ki values are in the small range of 1.8 - 2.6 microM, indicating that the short substituted group at the 2-position (OH; O-acetyl; NH2; N-acetyl) of the long chain octadecanol part of the phosphodiesters exhibit no essential role on the strong inhibitory potency of these alkylphosphocholines on the 7-ethoxycoumarin-O-deethylase activity.     

Reconstitution of the Z-Disk

Methanol-utilizing bacteria, Klebsiella sp. No. 101 and Microcyclus eburneus could grow aerobically and statically on 1,2-propanediol. The authors examined the presence of enzyme activity of adenosyl-B₁₂ dependent diol dehydratase as well as NAD dependent diol dehydroagenase. Adenosyl-B₁₂ dependent diol dehydratase activity was not detected in these organisms, even if these grown statically.

The dehydrogenase activity was found in the extract from these methanol-utilizing bacteria cells grown on various carbon sources. The partially purified enzyme preparation from the cells of Mic. eburneus grown aerobically on 1,2-propanediol dehydrogenated 1,2-propanediol, 1,2-butanediol and 2,3-butanediol. The enzyme activity was separated into two fractions, namely enzyme I and II on DEAE-Sephadex A-25 column chromatography. The enzyme I was different from the enzyme II in the ratio of enzyme activity to 1,2-propanediol and 2,3-butanediol, heat stability, pH stability and pH optimum, and effect of 2-mercaptoethanol.     

Glycerol and propanediols degradation by Desulfovibrio alcoholovorans in pure culture in the presence of sulfate, or in syntrophic association with Methanospirillum hungatei

Abstract In a mineral medium containing sulfate as terminal electron acceptor, the sulfate-reducing bacterium Desulfovibrio alcoholovorans oxidized stoichiometrically 1 mol glycerol to 1 mol acetate and 1 mol 1,3-propanediol to 1 mol acetate with the concomitant reduction of 0.75 and 1 mol sulfate, respectively; 1 mol 1,2-propanediol was degraded to 0.8 mol acetate and 0.1 mol proprionate, with the reduction of approximately 1 mol sulfate. The maximum specific growth rates (μ_max in h⁻¹) were 0.22, 0.086 and 0.09 with glycerol, 1,3-propanediol and 1,2-propanediol, respectively. The growth yields were 12.7 g, 11.1 g and 7.2 g dry weight/mol 1,3-propanediol, glycerol and 1,2-propanediol degraded, respectively. The growth yields and maximum specific growth rates of the H₂-transferring associations were also calculated. In the absense of sulfate, all these reduced substrates were degraded to acids and methane when D. alcoholovorans was cocultured with Methanospirillum hungatei . Changes in the metabolic pathway were observed in the degradation of 1,2- and 1,3-propanediol. The metabolic efficiency of D. alcoholovorans to degrade glycerol, 1.2- and 1,3-propanediol is discussed.