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

ABSTRACT: 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.

SUBMITTER: Tuner JM

PROVIDER: S-EPMC1265012 | biostudies-other | 1966 May

REPOSITORIES: biostudies-other

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Project description: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.

Project description:1. Pseudomonas sp. N.C.I.B. 8858 grew well on d- and l-1-aminopropan-2-ol and on aminoacetone. 2. Cell-free extracts possessed high activities of inducibly formed l-1-aminopropan-2-ol-NAD(+) oxidoreductase, amino alcohol-ATP phosphotransferase, dl-1-aminopropan-2-ol O-phosphate phospho-lyase and aldehyde-NAD(+) oxidoreductase, but no 1-aminopropan-2-ol racemase or d-1-aminopropan-2-ol-NAD(+) oxidoreductase. 3. The amino alcohol kinase (activated by ADP) was non-stereospecific towards 1-aminopropan-2-ol and was one-third as active with ethanolamine. The phospho-lyase was active with l- and d-1-aminopropan-2-ol O-phosphate, but ethanolamine O-phosphate was only one-tenth as active as its higher homologues. The purified aldehyde dehydrogenase was active with propionaldehyde, acetaldehyde and also with methylglyoxal. The previously observed 2-oxo aldehyde dehydrogenase activity was considered to be due to the broadly specific aldehyde dehydrogenase. 4. Mutants of Pseudomonas sp. N.C.I.B. 8858 deficient in 1-aminopropan-2-ol kinase, 1-aminopropan-2-ol O-phosphate phospho-lyase, aldehyde dehydrogenase or an enzyme involved in propionate metabolism were incapable of growth on aminoacetone or 1-aminopropan-2-ol as carbon source, although all except the kinase- or phospho-lyasedeficient mutants could use these compounds and ethanolamine as nitrogen sources. The aldehyde dehydrogenase-deficient mutants produced copious amounts of propionaldehyde and acetaldehyde during growth on the corresponding amino alcohols. 5. The path of aminoacetone metabolism in Pseudomonas sp. N.C.I.B. 8858 was concluded to involve l-1-aminopropan-2-ol, the O-phosphate ester of this compound, propionaldehyde and propionate as obligatory intermediates. d-1-Aminopropan-2-ol was metabolized by the same route as the l-isomer, gratuitously inducing formation of the stereospecific l-1-aminopropan-2-ol dehydrogenase. 6. Extracts of the pseudomonad grown with ethanolamine as the nitrogen source were devoid of 1-aminopropan-2-ol dehydrogenase, the kinase and the phospho-lyase, but exhibited cobamide coenzyme-dependent deaminase activity. Mutants deficient in kinase or phospho-lyase (deaminating) grew well on ethanolamine as the nitrogen source. Ethanolamine deaminase was inactive with, but inhibited by, 1-aminopropan-2-ol.

Project description: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.

Project description:Bacterial microcompartments encapsulate enzymatic pathways that generate small, volatile, aldehyde intermediates. The Rhodococcus and Mycobacterium microcompartment (RMM) operon from Mycobacterium smegmatis encodes four enzymes, including (S)-1-amino-2-propanol dehydrogenase and a likely propionaldehyde dehydrogenase. We show here that a third enzyme (and its nonmicrocompartment-associated paralog) is a moderately specific (S)-1-amino-2-propanol kinase. We determined the structure of apo-aminopropanol kinase at 1.35 Å, revealing that it has structural similarity to hexosamine kinases, choline kinases, and aminoglycoside phosphotransferases. We modeled substrate binding, and tested our model by characterizing key enzyme variants. Bioinformatics analysis established that this enzyme is widespread in Actinobacteria, Proteobacteria, and Firmicutes, and is very commonly associated with a candidate phospholyase. In Rhizobia, aminopropanol kinase is generally associated with aromatic degradation pathways. In the RMM (and the parallel pathway that includes the second paralog), aminopropanol kinase likely degrades aminoacetone through a propanolamine-phosphate phospho-lyase-dependent pathway. These enzymatic activities were originally described in Pseudomonas, but the proteins responsible have not been previously identified. Bacterial microcompartments typically co-encapsulate enzymes which can regenerate required co-factors, but the RMM enzymes require four biochemically distinct co-factors with no overlap. This suggests that either the RMM shell can uniquely transport multiple co-factors in stoichiometric quantities, or that all enzymes except the phospho-lyase reside outside of the shell. In summary, aminopropanol kinase is a novel enzyme found in diverse bacteria and multiple metabolic pathways; its presence in the RMM implies that this microcompartment degrades aminoacetone, using a pathway that appears to violate some established precepts as to how microcompartments function.

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

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