Bacterial metabolism of propane-1,2-diol

The pathway of propane-1,2-diol metabolism by a species of Flavobacterium able to grow on the diol as the sole source of carbon was influenced by the degree of aeration of the growth medium. Under strongly aerobic conditions the diol was exclusively catabolised to lactaldehyde by an initial diol oxidase, subsequently metabolised to pyruvate and then oxidised to CO₂ by the tricarboxylic acid cyle. Under microaerophilic conditions some propane-1,2-diol was catabolised by the oxidase-initiated pathway, but some diol was alternatively catabolised by an inducible diol dehydrase to propionaldehyde and subsequently reduced to n-propanol as an end product of metabolism.     

Bacterial metabolism of propane-1,2-diol

The pathway of propane-1,2-diol metabolism by a species of Flavobacterium able to grow on the diol as the sole source of carbon was influenced by the degree of aeration of the growth medium. Under strongly aerobic conditions the diol was exclusively catabolised to lactaldehyde by an initial diol oxidase, subsequently metabolised to pyruvate and then oxidised to CO2 by the tricarboxylic acid cycle. Under microaerophilic conditions some propane-1,2-diol was catabolised by the oxidase-initiated pathway, but some diol was alternatively catabolised by an inducible diol dehydrase to propionaldehyde and subsequently reduced to n-propanol as an end product of metabolism.     

Microbial metabolism of aliphatic glycols bacterial metabolism of ethylene glycol

A species of Flavobacterium isolated from pond water by its ability to grow aerobically on ethylene glycol as the role source of carbon initially oxidised the diol to glyoxylate via glycollate. The glyoxylate was metabolised by the glycerate pathway to acetyl-CoA. The acetyl-CoA was further metabolised by the tricarboxylic acid cycle plus malate synthase acting anaplerotically.     

Microbial metabolism of aliphatic glycols. Bacterial metabolism of ethylene glycol

A species of Flavobacterium isolated from pond water by its ability to grow aerobically on ethylene glycol as the role source of carbon initially oxidised the diol to glyoxylate via glycollate. The glyoxylate was metabolised by the glycerate pathway to acetyl-CoA. The acetyl-CoA was further metabolised by the tricarboxylic acid cycle plus malate synthase acting anaplerotically.     

Pathways of Anaerobic Acetate Utilization in Escherichia coli and Aerobacter cloacae

Acetate-1-¹⁴C was added to anaerobic glucose-fermenting cultures of Escherichia coli and Aerobacter cloacae. In the E. coli culture, lactate formation occurred late in the fermentation, when the rate of production of formate and acetate had decreased. The occurrence of acetate label in the lactate indicated formation of pyruvate from acetyl-coenzyme A (CoA) and formate. In the A. cloacae cultures, substantial amounts of acetate label were found in the 2,3-butanediol formed. Evidence is presented that the label could have entered the diol only by conversion of formate and acetyl-CoA into pyruvate. The observed levels of radioactivity in the diol indicated that during diol formation the reaction yielding formate and acetyl-CoA from pyruvate CoA was operating close to equilibrium. The shift in metabolism from formation of acetate, ethyl alcohol, and formate to the formation of butanediol or lactate appears to be due basically to an approach to equilibrium of the pyruvate-splitting reaction, whatever the induction mechanism by which the shift is implemented.     

Metabolism of threonine by Fusaria growth on threonine as the sole carbon and nitrogen source

Three strains ofFusarium supporting aerobic growth onl-threonine as the sole source of energy and carbon and nitrogen, initially metabolised threonine to acetyl-CoA and glycine via induciblel-threonine:NAD⁺ dehydrogenase plus 2-amino-3-oxobutyrate:CoA ligase activities. Comparative enzyme induction patterns after growth of the three strains on a wide range of carbon sources indicated that the glycine produced by the NAD⁺ plus CoASH-dependent cleavage of threonine was subsequently utilised as an energy source and biosynthetic precursor via the glycine-serine pathway, pyruvate carboxylase, and ultimately the TCA cycle. Acetyl-CoA, the second initial C₂ threonine catabolism product, was subsequently assimilated via a combined TCA plus glyoxylate cycle.     

Pathways of acetate and propionate production in adult Fasciola hepatica

In adult F. hepatica pyruvate is decarboxylated via pyruvate dehydrogenase to acetyl-CoA; acetyl-CoA is then cleaved to acetate via three possible mechanisms (1) carnitine dependent hydrolysis, (2) CoA transferase, (3) reversal of a GTP dependent acyl-CoA synthetase. Of these three systems, CoA transferase has by far the greatest activity. Propionate production by F. hepatica is similar to the mammalian system, succinate being metabolized via succinic thiokinase, methylmalonyl-CoA isomerase, methyl-malonyl-CoA racemase and propionyl-CoA carboxylase to propionyl-CoA. Propionyl-CoA is then cleaved to propionate by the same three pathways as acetyl-CoA. No ATP or GTP production could be demonstrated when acetyl- or propionyl-CoA were incubated with homogenates of F. hepatica. This indicates that carnitine dependent hydrolysis or CoA transferase are the major pathways of acetyl- or propionyl-CoA breakdown. The CoA transferase reaction would result in the conservation of the bond energy although there is no net ATP synthesis.     

Ethylene Glycol Metabolism by Pseudomonas putida

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.     

New evidence for the mechanism of the tin(II) chloride catalyzed reactions of vicinal diols with diazodiphenylmethane in 1,2-dimethoxyethane

A kinetic study of the tin(II) chloride catalyzed reaction of diazodiphenylmethane with ethylene glycol in dimethoxyethane is reported. The preparation and characterization of ethylene glycol monodiphenylmethyl ether, the main product from this reaction, is also reported as well as the preparation of the two diphenylmethyl monoethers of methyl 4,6-O-benzylidene-alpha-D-glucopyranoside. An unexpected relationship between the concentration of ethylene glycol and the pseudo first-order rate constant, k', was observed in these reactions. For low concentrations of ethylene glycol (below 0.06 M), k' increases with increasing concentration of the diol. This trend is reversed for high concentrations of ethylene glycol (from about 0.06 to about 0.2 M). The apparent rate constant was also inversely related to the initial concentration of diazodiphenylmethane for the concentrations investigated. These results make the previously proposed involvement of a 1,3,2-dioxastannolane intermediate very unlikely [Petursson, S.; Webber, J.M. Carbohydr. Res. 1982, 103, 41-52]. The results suggest that more likely intermediates for these reactions involve tin(II) chloride complexes in a dynamic equilibrium with the diol.     

Influence of isoform and DNP on butyrate transport across the sheep ruminal epithelium

Short-chain fatty acids are absorbed in considerable amounts from the rumen. During transit through the epithelial layer, they are intensively metabolised. Interaction between intraepithelial metabolism and absorption, however, is hardly understood. The present study therefore compared the transepithelial transport of the easily metabolised n-butyrate with that of the more metabolism-resistant iso-butyrate both under in vivo conditions (isolated and washed reticulorumen) and in vitro conditions (Ussing chamber). Under in vivo conditions, net absorption of n-butyrate was significantly higher than that of iso-butyrate. The in vitro experiments showed that the higher net flux of n-butyrate was solely due to a higher mucosal-to-serosal flux, whereas the serosal-to-mucosal flux of butyrate was independent from the isoform. Blocking intraepithelial ATP delivery by 2,4-dinitrophenol abolished the net flux of n-butyrate. The study indicates that metabolism and/ or ATP availability stimulates n-butyrate net absorption. By this, the metabolic activity of the epithelium may have a regulatory influence on absorption of n-butyrate.     

Toxicity of cryoprotectants to honey bee semen and queens

Given the threats to the intraspecific biodiversity of Apis mellifera and the pressure on bee breeding to come up with disease-tolerant lines, techniques to cryopreserve drone semen are of great interest. Freeze-thawed drone semen of high viability and/or motility has repeatedly been obtained, but fertility of such semen, when it was measured, was always low. The cryoprotective agent (CPA) most frequently used with drone semen is dimethyl sulfoxide (DMSO), although this substance has been suspected of causing genetic damage in sperm. No form of sperm washing is currently performed. Using a membrane permeability assay, we measured the short-term toxicity of four possible replacements for DMSO, 1,3-propane diol, 2,3-butane diol, ethylene glycol, and dimethyl formamide. We also tested whether the practice of inseminating queens with CPA-containing semen affects sperm numbers in the storage organs of queens, or sperm fertility. Finally, we tested whether CPA-toxicity in vivo can be reduced by using mixtures of two CPAs, DMSO, and ethylene glycol. Our results show that, although short-term toxicity of all CPAs tested was low, the presence of single CPAs in insemination mixtures at concentrations required for slow freezing greatly reduced the number of sperm reaching the spermatheca. Contrary to earlier reports, this was also true for DMSO. Ethylene glycol was additionally shown to reduce the viability of spermatozoa reaching the storage organ. Mixtures of DMSO and EthGly performed better than either substance used singly at the same concentration. We conclude that the toxicity of CPAs, including DMSO, on honey bee semen and/or queens has been underestimated in the past. This could partly explain the discrepancy between in vitro and in vivo quality of cryopreserved drone semen, described by others. Combinations of several CPAs and techniques to partly remove CPAs after thawing could help to solve this problem.     

Enhancement of the Oral Bioavailability of Felodipine Employing 8-Arm-Poly(Ethylene Glycol): <Emphasis Type="Italic">In Vivo</Emphasis>, <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Silico</Emphasis> Evaluation

Poor oral bioavailability is the single most important challenge in drug delivery. Prominent among the factors responsible for this is metabolic activity of the intestinal and hepatic cytochrome P450 (CYP450) enzymes. In preliminary studies, it was demonstrated that 8-arm-PEG was able to inhibit the felodipine metabolism. Therefore, this report investigated the oral bioavailability-enhancing property of 8-arm-PEG employing detailed in vitro, in vivo, and in silico evaluations. The in vitro metabolism of felodipine by cytochrome P450 3A4-expressed human liver microsomes (HLM) was optimized yielding a typical Michaelis–Menten plot through the application of Enzyme Kinetic Module software from where the enzyme kinetic parameters were determined. In vitro investigation of 8-arm-poly(ethylene glycol) against CYP3A4-catalyzed felodipine metabolism employing human liver microsomes compared closely with naringenin, a typical grapefruit flavonoid, yielding IC₅₀ values of 7.22 and 121.97 μM, respectively. The investigated potential of 8-arm-poly(ethylene glycol) in oral drug delivery yielded satisfactory in vitro drug release results. The in vivo studies of the effects of 8-arm-poly(ethylene glycol) on the oral bioavailability of felodipine as performed in the Large White pig model showed a >100% increase in plasma felodipine levels compared to controls, with no apparent effect on systemic felodipine clearance. The outcome of this research presents a novel CYP3A4 inhibitor, 8-arm-poly(ethylene glycol) for oral bioavailability enhancement.     

Phosphoinositide inhibition of acetyl-CoA carboxylase from rat liver

When purified acetyl-CoA carboxylase was incubated with various phospholipids, the effects on carboxylase activity were quite diverse. Phosphatidic acid, phosphatidylcholine, and phosphatidylinositol were slightly stimulatory, whereas carboxylase was inhibited by polyphosphoinositides in a time- and concentration-dependent manner. Phosphatidylinositol 4,5-bisphosphate (TPI) was the most effective inhibitor; carboxylase activity was inhibited 50% after incubation with 1.5 μm TPI for 30 min. Incubation of carboxylase with citrate reduced the susceptibility to inhibition by TPI. The inhibition was reversed by removal of TPI from the inhibited enzyme. Incubation of TPI with divalent metal cations removed its ability to inhibit carboxylase. Sedimentation studies showed that TPI treatment shifts carboxylase to a less-polymerized form. The K_m for ATP, 24 μm, was not affected by the inhibitor. However, the apparent K_m for acetyl-CoA was decreased from 44 to 11 μm following incubation with TPI. The possibility that polyphosphoinositides may play a role in acetyl-CoA carboxylase regulation is discussed.     

What factors are responsible for the greater yield of ATP per carbon atom when fatty acids are completely oxidised to CO2 and water compared with glucose?

Some current textbooks of biochemistry present calculations which indicate that when palmitic or stearic acid is completely oxidised to CO₂ and water, more molecules of ATP are produced per carbon atom than when glucose is similarly oxidised. This greater yield of ATP is not due to ATP produced as a result of the β-oxidative process, but rather due to the increased yield of acetyl-CoA molecules produced from fatty acids (3 acetyl-CoA molecules per 6 carbon atoms) compared with that produced from glucose (2 acetyl-CoA molecules per 6 carbon atoms).     

Metabolism of methanogens

Methanogenic archaea convert a few simple compounds such as H₂ + CO₂, formate, methanol, methylamines, and acetate to methane. Methanogenesis from all these substrates requires a number of unique coenzymes, some of which are exclusively found in methanogens. H₂-dependent CO₂ reduction proceeds via carrier-bound C₁ intermediates which become stepwise reduced to methane. Methane formation from methanol and methylamines involves the disproportionation of the methyl groups. Part of the methyl groups are oxidized to CO₂, and the reducing equivalents thereby gained are subsequently used to reduce other methyl groups to methane. This process involves the same C₁ intermediates that are formed during methanogenesis from CO₂. Conversion of acetate to methane and carbon dioxide is preceeded by its activation to acetyl-CoA. Cleavage of the latter compound yields a coenzyme-bound methyl moiety and an enzyme-bound carbonyl group. The reducing equivalents gained by oxidation of the carbonyl group to carbon dioxide are subsequently used to reduce the methyl moiety to methane. All these processes lead to the generation of transmembrane ion gradients which fuel ATP synthesis via one or two types of ATP synthases. The synthesis of cellular building blocks starts with the central anabolic intermediate acetyl-CoA which, in autotrophic methanogens, is synthesized from two molecules of CO₂ in a linear pathway.     

Acetyl-CoA synthetase (ADP forming) in archaea,a novel enzyme involved in acetate formation and ATP synthesis

The anaerobic hyperthermophilic archaea Desulfurococcus amylolyticus, Hyperthermus butylicus, Thermococcus celer, Pyrococcus woesei, the hyperthermophilic bacteria Thermotoga maritima and Clostridium thermohydrosulfuricum and the aerobic mesophilic archaeon Halobacterium saccharovorum were grown either on complex media, on sugars or on pyruvate as carbon and energy sources. During growth acetate was formed as fermentation product by all organisms. The enzymes involved in acetyl-CoA formation from pyruvate and in acetate formation from acetyl-CoA were investigated:

1. Cell extracts of all species, both archaea and bacteria, catalyzed the coenzyme A-dependent oxidative decarboxylation of pyruvate with viologen dyes or with Clostridium pasteurianum ferredoxin as electron acceptors indicating a pyruvate: ferredoxin oxidoreductase to be operative in acetyl-CoA formation from pyruvate.
2. Cell extracts of all archaeal species, both hyperthermophiles (D. amylolyticus, H. butylicus, T. celer, P. woesei) and the mesophile H. saccharovorum, contained an acetyl-CoA synthetase (ADP forming), which catalyzes both acetate formation from acetyl-CoA and ATP synthesis from ADP and phosphate (P_i): Acetyl-CoA+ADP+P_i?Acetate + ATP+CoA. Phosphate acetyltransferase and acetate kinase could not be detected.
3. Cell extracts of the hyperthermophilic (eu)bacteria T. maritima and C. thermohydrosulfuricum contained phosphate acetyltransferase and acetate kinase rather than acetyl-CoA synthetase (ADP forming).

These data indicate that acetyl-CoA synthetase (ADP forming) represents a typical archaeal property rather than an enzyme specific for hyperthermophiles. It is proposed that in all acetate forming archaea the formation of acetate and of ATP from acetyl-CoA, ADP and P_i are catalyzed by acetyl-CoA synthetase (ADP forming), whereas in all acetate forming (eu)bacteria these reactions are catalyzed by two enzymes, phosphate acetyltransferase and acetate kinase.     

Regulation of rat brainstem tryptophan 5-monooxygenase. Calcium-dependent reversible activation by ATP and magnesium.

Tryptophan 5-monooxygenase in rat brainstem cytosol was activated about twofold by incubation with 0.5 mm ATP and 5 mm MgCl₂. The activation required micromolar concentrations of Ca²⁺ but was not dependent on either cyclic AMP or cyclic GMP. Rat brain cytosol was shown to possess an endogenous protein kinase which was markedly stimulated by the addition of Ca²⁺ using endogenous protein substrates. Following activation by ATP and Mg²⁺ in the presence of Ca²⁺, tryptophan 5-monooxygenase was reversibly deactivated to the original level by incubation at 30 °C after removal of Ca²⁺ by adding ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid and was then reactivated by incubation at 30 °C after subsequent addition of Ca²⁺ and ATP. The deactivation was markedly inhibited by the omission of Mg²⁺ or by the addition of NaF.     

Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica

Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PP_i-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PP_i were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PP_i displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.