Microbial metabolism of amino ketones. Aminoacetone formation from 1-aminopropan-2-ol by a dehydrogenase in Escherichia coli

1. Washed-cell suspensions of Escherichia coli, incubated at the optimum pH of 6.4 and with a saturating substrate concentration of approx. 10mm, convert dl-1-aminopropan-2-ol into aminoacetone at a rate of approx. 4.0mmumoles/mg. dry wt. of cells/min. at 30 degrees . 2. Mg(2+), Mn(2+), Co(2+), Zn(2+), Ca(2+), K(+) and NH(4) (+), as sulphates, and EDTA have no effect on this rate, although Cu(2+) inhibits and Fe(2+) activates to some extent. 3. Conditions of growth markedly affect the rate of aminoacetone production by cell suspensions. 4. Dialysed cell-free extracts of E. coli exhibit 1-aminopropan-2-ol-dehydrogenase activity, the enzyme having optimum activity at pH7.0, a requirement for NAD(+) and K(+), and a K(m) for the amino alcohol substrate of 0.8mm, calculated for a single enantiomorph. 5. Under optimum conditions 1-aminopropan-2-ol dehydrogenase forms aminoacetone at rate of approx. 3.0mmumoles/mg. of protein/min. at 37 degrees . The enzyme is only slightly inhibited by dl-3-hydroxybutyrate and dl-2-hydroxy-2-phenylethyl-amine. 6. l-Threonine-dehydrogenase activity is exhibited by both whole cells and cell-free extracts. Whole cells produce aminoacetone from l-threonine more slowly than they do from dl-1-aminopropan-2-ol, whereas the situation is reversed in cell-free extracts. Both kinetic evidence, and the fact that synthesis of 1-aminopropan-2-ol dehydrogenase, but not of threonine dehydrogenase, is repressed by compounds such as glucose and pyruvate, provide evidence that the amino alcohol is oxidized by a specific enyme. 7. The metabolic role of 1-aminopropan-2-ol dehydrogenase is discussed.     

Microbial metabolism of amino alcohols. 1-Aminopropan-2-ol and ethanolamine metabolism via propionaldehyde and acetaldehyde in a species of Pseudomonas

1. Growth and manometric experiments showed that a Pseudomonas sp. P6 (N.C.I.B. 10431), formerly known as Achromobacter sp. P6, was capable of growth on both stereoisomers of 1-aminopropan-2-ol, and supported the hypothesis that assimilation involved metabolism to propionaldehyde, propionate and possibly 2-hydroxyglutarate. A number of alternative intermediary metabolites were ruled out. 2. Accumulation of propionaldehyde from 1-aminopropan-2-ol by intact cells occurred only during active growth, was transitory and was accompanied by morphological changes in the pseudomonad. 3. Enzymic and radioactive tracer evidence showed that 1-aminopropan-2-ol O-phosphate was the intermediate between amino alcohol and aldehyde. The operation of an inducibly formed ATP-amino alcohol phosphotransferase was established by measuring substrate disappearance, ADP formation and amino alcohol O-phosphate formation. This novel kinase had two activity peaks at about pH7 and 9. It acted on both l- and d-isomers of 1-aminopropan-2-ol, and also on l-threonine and ethanolamine, but had only low activity towards choline. The enzyme was partially purified by ion-exchange chromatography. 4. An amino alcohol O-phosphate phospho-lyase (deaminating) produced propionaldehyde from dl- and d-1-aminopropan-2-ol O-phosphate, and also formed acetaldehyde less rapidly from ethanolamine O-phosphate. It had optimum activity at about pH8 in Tris-HCl buffers. The enzyme was partially purified and evidence was obtained that a single enzyme was responsible for both activities. Apparent K(m) values for the substrates were determined. Activity was inhibited by dl-threonine O-phosphate, dl-serine O-phosphate, choline O-phosphate and P(i). Enzyme formation was induced by growth with either amino alcohol substrate. 5. Radioactive tracer experiments with dl-1-amino[3-(14)C]propan-2-ol confirmed the operation of the amino alcohol kinase and demonstrated coupling with the phospho-lyase enzyme in vitro to produce [(14)C]-propionaldehyde. 6. An aldehyde dehydrogenase, found in extracts of the pseudomonad after growth on 1-aminopropan-2-ol, was characterized and concluded to be responsible for propionaldehyde and acetaldehyde oxidation. The enzyme was inactive with methylglyoxal. 7. Propionate and acetate were concluded to be metabolized via propionyl-CoA and acetyl-CoA, and studies were made of a CoA ester synthase found in extracts. 8. Studies of a strain of Pseudomonas putida N.C.I.B. 10558 suggested that 1-aminopropan-2-ols were metabolized via their O-phosphates, propionaldehyde and propionate. Amino alcohol kinase activity was detected and extracts contained a phospho-lyase showing higher activity with the 1-aminopropan-2-ol O-phosphate than with ethanolamine O-phosphate.     

Microbial metabolism of amino alcohols. Metabolism of ethanolamine and 1-aminopropan-2-ol in species of Erwinia and the roles of amino alcohol kinase and amino alcohol O-phosphate phospho-lyase in aldehyde formation

1. Growth of Erwinia carotovora N.C.P.P.B. 1280 on media containing 1-aminopropan-2-ol compounds or ethanolamine as the sole N source resulted in the excretion of propionaldehyde or acetaldehyde respectively. The inclusion of (NH(4))(2)SO(4) in media prevented aldehyde formation. 2. Growth, microrespirometric and enzymic evidence implicated amino alcohol O-phosphates as aldehyde precursors. An inducibly formed ATP-amino alcohol phosphotransferase was partially purified and found to be markedly stimulated by ADP, unaffected by NH(4) (+) ions and more active with ethanolamine than with 1-aminopropan-2-ol compounds. Amino alcohol O-phosphates were deaminated by an inducible phospho-lyase to give the corresponding aldehydes. This enzyme, separated from the kinase during purification, was more active with ethanolamine O-phosphate than with 1-aminopropan-2-ol O-phosphates. Activity of the phospho-lyase was unaffected by a number of possible effectors, including NH(4) (+) ions, but its formation was repressed by the addition of (NH(4))(2)SO(4) to growth media. 3. E. carotovora was unable to grow with ethanolamine or 1-aminopropan-2-ol compounds as sources of C, the production of aldehydes during utilization as N sources being attributable to the inability of the microbe to synthesize aldehyde dehydrogenase. 4. Of seven additional strains of Erwinia examined similar results were obtained only with Erwinia ananas (N.C.P.P.B. 441) and Erwinia milletiae (N.C.P.P.B. 955).     

The phosphorylation and subsequent metabolism of 1-aminopropan-2-ol

Rat liver mitochondria contain an apparently substrate-specific 1-aminopropan-2-ol kinase activity. Indirect evidence also indicates the presence of a phosphoryl-1-aminopropan-2-ol cytidylyl transferase activity. A possible role for these two enzymes in the incorporation of 1-aminopropan-2-ol as a phospholipid base is considered in the light of this and other data.     

The metabolism of cyclopentanol by Pseudomonas N.C.I.B. 9872

1. Pseudomonas N.C.I.B. 9872 grown on cyclopentanol as carbon source oxidized it at a rate of 228mul of O(2)/h per mg dry wt. and the overall consumption of 5.9mumol of O(2)/mumol of substrate. Cyclopentanone was oxidized at a similar rate with the overall consumption of 5.2mumol of O(2)mumol of substrate. Cells grown with sodium acetate as sole source of carbon were incapable of significant immediate oxidation of these two substrates. 2. Disrupted cells catalysed the oxidation of cyclopentanol to cyclopentanone by the action of an NAD(+)-linked dehydrogenase with an alkaline pH optimum. 3. A cyclopentanolinduced cyclopentanone oxygenase (specific activity 0.11mumol of NADPH oxidized/min per mg of protein) catalysed the consumption of 1mumol of NADPH and 0.9mumol of O(2) in the presence of 1mumol of cyclopentanone. NADPH oxidation did not occur under anaerobic conditions. The only detectable reaction product with 100000g supernatant was 5-hydroxyvalerate. 4. Extracts of cyclopentanol-grown cells contained a lactone hydrolase (specific activity 7.0mumol hydrolysed/min per mg of protein) that converted 5-valerolactone into 5-hydroxyvalerate. 5. Cyclopentanone oxygenase fractions obtained from a DEAE-cellulose column were almost devoid of 5-valerolactone hydrolase and catalysed the formation of 5-valerolactone in high yield from cyclopentanone in the presence of NADPH. 6. Incubation of 5-hydroxyvalerate with the 100000g supernatant, NAD(+) and NADP(+) under aerobic conditions resulted in the consumption of O(2) and the conversion of 5-hydroxyvalerate into glutarate. 7. The high activity of isocitrate lyase in cyclopentanol-grown cells suggests that the further oxidation of glutarate proceeds through as yet uncharacterized reactions to acetyl-CoA. 8. The reaction sequence for the oxidation of cyclopentanol by Pseudomonas N.C.I.B. 9872 is: cyclopentanol --> cyclopentanone --> 5-valerolactone --> 5-hydroxyvalerate --> glutarate --> --> acetyl-CoA.     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590.

The microbial degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 was studied, and two major products were isolated and identified as 7 alpha, 12 beta-dihydroxyandrosta-1,4-diene-3,17-dione and 7 alpha, 12 alpha-dihydroxy-3-oxopregna-1,4-diene-20-carboxylic acid. Four minor products were isolated and evidence is given for the following structures: 7 alpha, 12 alpha-dihydroxyandrosta-1,4-diene-3,17-dione, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 7 alpha, 12 beta, 17 beta-trihydroxyandrosta-1,4-dien-3-one and 7 alpha, 12 alpha-dihydroxy-3-oxopregn-4-ene-20-carboxylic acid. The significance of the production of the steroid products is discussed, along with the possible enzymic mechanisms responsible for their production.     

Microbial metabolism of amino ketones: l-1-Aminopropan-2-ol dehydrogenase and l-threonine dehydrogenase in Escherichia coli

1. A wide range of intermediary metabolites and substrate analogues have no effect on the oxidation of dl-1-aminopropan-2-ol to aminoacetone by washed-cell suspensions of Escherichia coli. Only dl-2-hydroxy-2-phenylethylamine, dl-1,3-diaminopropan-2-ol, dl-serine and l-1-(3,4-dihydroxyphenyl)-2-aminoethanol act as inhibitors. 2. Dialysed cell-free extracts of E. coli exhibit an NAD(+)-dependent dl-1-aminopropan-2-ol-dehydrogenase activity of approx. 8mmumoles of aminoacetone formed/mg. of protein/min. at the pH optimum of approx. 10. The K(m) values for the coenzyme and dl-amino alcohol are approx. 0.4 and 10.0mm respectively. A smaller peak of activity occurs at pH7.0-7.2, the K(m) for NAD(+) at pH7 being approx. 0.05mm. 3. Enzyme activity in cell-free extracts is inhibited by dl-2-hydroxy-2-phenylethylamine, dl-1-aminopropane-2,3-diol and dl-serine. dl-Phenylserine and dl-1-aminobutan-2-ol are oxidized to compounds reacting as amino ketones. 4. In fresh cell-free extracts l(+)-1-aminopropan-2-ol preparations are oxidized more rapidly than racemic or laevo-rotatory material, the d(-)-enantiomorph appearing to act as a competitive inhibitor. The K(m) for l(+)-1-aminopropan-2-ol appears to be approx. 1.5mm when highly resolved substrate preparations are used, either in the free base form or as the l(+)-tartrate salt. 5. l(+)-1-Aminopropan-2-ol dehydrogenase is a labile enzyme, and in appropriately treated extracts activity towards the d-enantiomorph is detectable and relatively higher than that towards the l-enantiomorph. 6. Optimum activity of l-threonine-dehydrogenase in cell-free extracts is exhibited at pH9.6 in the presence of NAD(+). The K(m) values for coenzyme and amino acid substrate are approx. 0.08 and 5.0mm respectively. This enzyme is distinct from 1-aminopropan-2-ol dehydrogenases on the basis of kinetic evidence, and the separation of activities by gel filtration. 7. Both l-threonine and dl-1-aminopropan-2-ol dehydrogenases are markedly inhibited by 8-hydroxyquinoline and p-chloromercuribenzoate, but only slightly by other chelating and thiol reagents. 8. E. coli is incapable of growth on simple synthetic media, containing a variety of carbon sources, when dl-1-aminopropan-2-ol is supplied as the sole source of nitrogen. It appears unlikely that the micro-organism can deaminate aminoacetone. 9. The metabolic roles of l-threonine dehydrogenase, aminoacetone and 1-aminopropan-2-ol dehydrogenases are discussed.     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 under anaerobic conditions.

The bacterial degradation of cholic acid under anaerobic conditions by Pseudomonas sp. N.C.I.B. 10590 was studied. The major unsaturated neutral compound was identified as 12 beta-hydroxyandrosta-4,6-diene-3,17-dione, and the major unsaturated acidic metabolite was identified as 12 alpha-hydroxy-3-oxochola-4,6-dien-24-oic acid. Eight minor unsaturated metabolites were isolated and evidence is given for the following structures: 12 alpha-hydroxyandrosta-4,6-diene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-4,6-dien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, 3,12-dioxochola-4,6-dien-24-oic acid and 12 alpha-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid. In addition, a major saturated neutral compound was isolated and identified as 3 beta,12 beta-dihydroxy-5 beta-androstan-17-one, and the only saturated acidic metabolite was 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid. Nine minor saturated neutral compounds were also isolated, and evidence is presented for the following structures: 12 beta-hydroxy-5 beta-androstane-3,17-dione, 12 alpha-hydroxy-5 beta-androstane-3,17-dione, 3 beta,12 alpha-dihydroxy-5 beta-androstan-17-one, 3 alpha,12 beta-androstan-17-one, 3 alpha,12 alpha-dihydroxy-5 beta-androstan-17-one, 5 beta-androstane-3 beta,12 beta,17 beta-triol, 5 beta-androstane-3 beta,12 alpha,17 beta-triol, 5 beta-androstane-3 alpha,12 beta,17 beta-triol and 5 beta-androstane-3 alpha,12 alpha,17 beta-triol. The induction of 7 alpha-dehydroxylase and 12 alpha-dehydroxylase enzymes is discussed, together with the significance of dehydrogenation and ring fission under anaerobic conditions.     

Microbial metabolism of amino alcohols. Purification and properties of coenzyme B12-dependent ethanolamine ammonia-lysae of Escherichia coli

1. The 120-fold purification of ethanolamine ammonia-lyase from Escherichia coli extracts, to apparent homogeneity, is described. Ethanolamine, dithiothreitol, glycerol and KCl protected the apoenzyme from inactivation. 2. At the optimum pH7.5, K(m) values for ethanolamine and coenzyme B(12) were 44mum and 0.42mum respectively. The K(m) for ethanolamine was markedly affected by pH, transitions occurring at pH7.0 and 8.35. 3. The enzyme was specific for ethanolamine as substrate, none of the 18 analogues tested being active. l-2-Aminopropan-l-ol (K(i) 0.86mum), dl-1-aminopropan-2-ol (K(i) 2.2mum) and dl-1,3-diaminopropan-2-ol (K(i) 88.0mum) inhibited competitively. 4. Enzyme activity was inhibited, irreversibly and non-competitively, by the coenzyme analogues methylcobalamin (K(i) 1.4nm), hydroxocobalamin (K(i) 2.1nm) and cyanocobalamin (K(i) 4.8nm). 5. Iodoacetamide inhibited in the absence of ethanolamine, but only slightly in its presence. p-Hydroxymercuribenzoate inhibited markedly even in the presence of ethanolamine. Dithiothreitol and 2-mercaptoethanol (less effectively) restored activity to the enzyme dialysed against buffer containing ethanolamine. 6. Although K(+) ions stabilized the enzyme during dialysis or storage, they were not necessary for activity. 7. Gel filtration showed the enzyme to be of high molecular weight, ultracentrifugal studies giving s(20,w) of 16.4 and an estimated mol.wt. 560400. The isoelectric point for the apoenzyme was approx. pH5.0. inhibited enzyme activity at concentrations above 1m (95% inhibition at 3m) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated protein subunits of mol.wt. 61400. 8. Immunological studies showed that the E.coli enzyme was closely related to those of other enterobacteria, but only distantly to that of Clostridium sp. A double precipitin band suggested that the apoenzyme may be made up of two protein components.     

Microbial sensors for naphthalene using Sphingomonas sp. B1 or Pseudomonas fluorescens WW4

 Amperometric biosensors for naphthalene were developed using either immobilized Sphingomonas sp. B1 or Pseudomonas fluorescens WW4 cells. The microorganisms were immobilized within a polyurethane-based hydrogel, which was used for a microbial biosensor for the first time. Both strains were shown to be equally suited for the quantification of naphthalene in aqueous solutions. The biosensors were tested in a flow-through system and a stirred cell (batch method). In both systems a linear response down to the detection limit was obtained. Measurements in the flow-through system gave sensitivities of up to 1.2 nA mg⁻¹ l⁻¹ and a linear range from 0.03 mg/l to 2.0 mg/l. The response time (t ₉₅) was 2 min and the sample throughput six per hour; the repeatability was within ±5 %. With the batch method, sensitivities of between 3 nA mg⁻¹ l⁻¹ and 5 nA mg⁻¹l⁻¹ and a linear range of 0.01–3.0 mg/l were obtained; the response time was between 3 min and 5 min. The sensors reached an operational lifetime of up to 20 days. The sensitivity of both sensors for naphthalene was, in most cases, more than four times higher than for various other substrates. Received: 18 October 1995/Received revision: 22 December 1995/Accepted: 22 January 1996     

Microbial metabolism of amino alcohols. Formation of coenzyme B12-dependent ethanolamine ammonia-lyase and its concerted induction in Escherichia coli.

1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.