Biomedical subjects

D H Pieper

Publications and source records attributed to D H Pieper.

28 records · Page 2Linked to original sources

PubMed1996

Muconolactone isomerase of the 3-oxoadipate pathway catalyzes dechlorination of 5-chloro-substituted muconolactones.

An enzyme of Alcaligenes eutrophus JMP 134 which catalyzes dechlorination of (4R, 5R)- and (4R,5S)-5-chloro-3-methyl- and (4R, 5S)-5-chloromuconolactone of principally 3-methyl-trans-, 3-methyl-cis-dienelactone and cis-dienelactone, respectively, was purified to homogeneity. The enzyme was identified as muconolactone isomerase on the basis of its high activity with muconolactone and on its high degree of sequence similarity with previously described muconolactone isomerases. Molecular mass determinations of the highly hydrophobic and heat-resistant enzyme indicated a decameric structure involving a single 10.100-kDa subunit similar to that of muconolactone isomerase of Pseudomonas putida. Kinetic analysis showed cooperative effects between the subunits during conversion of (4R, 5S)-5-chloro-3 -methylmuconolactone. (4R, 5S)-5-chloromuconolactone was the preferred substrate, over the natural substrate (4S)-muconolactone. The (4S, 5S)-structure was found to be an inhibitor of (4R, 5R)-5-chloro-3-methylmuconolactone transformation. Methylsubstitution of the substrate results in a higher affinity for the enzyme, but a drastically lower velocity, resulting in a lower specificity constant.

4-Butyrolactone↗

PubMed1996

Metabolism of 5-chlorosubstituted muconolactones.

The stereochemistry of the four stereoforms of 5-chloro-3-methylmuconolactones could be deduced from NMR and stability data, and from the comparison with authentic (4R, 5S)-5-chloromuconolactone. Muconolactone isomerase of Alcaligenes eutrophus JMP 134 was shown to catalyze syn-elimination of hydrogen chloride from (4R, 5R)-5-chloro-3-methylmuconolactone, (4R, 5S)-5-chloro-3 -methylmuconolactone and (4R, 5S)-5-chloromuconolactone to form 3-methyl-trans-dienelactone, 3-methyl-cis-dienelactone and a 3:1 mixture of cis- and trans-dienelactone, respectively. 3-Methyl-trans-dienelactone was a substrate of pJP4-encoded dienelactone hydrolase of A. eutrophus JMP 134, whereas 3-methyl-cis-dienelactone transformation was negligible indicating a restricted substrate specificity of this enzyme. Both substrates were transformed into 3-methylmaleylacetate which in turn was a substrate for maleylacetate reductase. This compound was shown to possess a cyclic structure (4-hydroxy-3-methyl-muconolactone) under acidic conditions.

4-Butyrolactone↗

PubMed1995

From xenobiotic to antibiotic, formation of protoanemonin from 4-chlorocatechol by enzymes of the 3-oxoadipate pathway.

Chloroaromatics, a major class of industrial pollutants, may be oxidatively metabolized to chlorocatechols by soil and water microorganisms that have evolved catabolic activities toward these xenobiotics. We show here that 4-chlorocatechol can be further transformed by enzymes of the ubiquitous 3-oxoadipate pathway. However, whereas chloromuconate cycloisomerases catalyze the dechlorination of 3-chloro-cis,cis-muconate to form cis-dienelactone, muconate cycloisomerases catalyze a novel reaction, i.e. the dechlorination and concomitant decarboxylation to form 4-methylenebut-2-en-4-olide (protoanemonin), an ordinarily plant-derived antibiotic that is toxic to microorganisms.

Adipates↗

PubMed1995

Formation of Dimethylmuconolactones from Dimethylphenols by Alcaligenes eutrophus JMP 134.

2,3-, 2,4-, 2,5-, 3,4-, and 3,5-dimethylphenols were cometabolized by 2,4-dichlorophenoxyacetate-grown Alcaligenes eutrophus JMP 134 or the constitutive derivative JMP 134-1 via the ortho pathway into dimethylmuconolactones as dead-end products. Formation of two distinct lactones from 3,4-dimethylphenol is indicative of 2- as well as 6-hydroxylation. Induction of the meta-cleavage pathway by 2,3- and 3,4-dimethylphenols resulted in growth and no accumulation of products. In contrast, 3,5-dimethylphenol is not metabolized by the meta-cleavage pathway.

Journal Article↗

PubMed1993

Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134.

2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 134-1. It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis,cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite. Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide. Those lactones harboring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1992

Degradation of 2,4-dinitrophenol by two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2.

Two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2, were isolated from soil and river water by their abilities to utilize 2,4-dinitrophenol (0.5 mM) as the sole source of nitrogen. Although succinate was supplied as a carbon and energy source during selection, both isolates could utilize 2,4-dinitrophenol also as the sole source of carbon. Both strains metabolized 2,4-dinitrophenol under concomitant liberation of stoichiometric amounts of nitrite and 4,6-dinitrohexanoate as a minor dead-end metabolite.

2,4-Dinitrophenol↗

PubMed1990

Purification and characterization of 4-methylmuconolactone methylisomerase, a novel enzyme of the modified 3-oxoadipate pathway in the gram-negative bacterium Alcaligenes eutrophus JMP 134.

4-Carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone) isomerase, transforming 4-methyl-2-enelactone to 3-methyl-2-enelactone, was purified from a derivative strain of Pseudomonas sp. B13, named B13 FR1, carrying the plasmid pFRC2OP. This plasmid contained the isomerase gene cloned from Alcaligenes eutrophus JMP 134, which uses 4-methyl-2-enelactone as a carbon source. The enzyme consists of a single peptide chain of Mr 40,000 as judged by SDS/PAGE. In addition to 4-methyl-2-enelactone, the putative reaction intermediate, 1-methyl-3,7-dioxo-2,6-dioxy-bicyclo[3.3.0]octane (1-methylbislactone), was a substrate for the enzyme, but kinetic data presented did not favour its role as a reaction intermediate. Isomeric methyl-substituted 4-carboxymethylbut-2-en-4-olides were neither substrates nor inhibitors. Possible reaction mechanisms are discussed.

Adipates↗

PubMed1990

Purification and characterization of dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134.

Dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134 was purified to homogeneity. The enzyme has an Mr of about 270,000 as determined by gel filtration and consists of six to eight subunits of identical Mr 40,000 as determined by SDS/PAGE. Mn2+ ions as well as thiol groups are required for activity. A high Km value of about 4 mM for cis,cis-muconate explains the reported low activity with this compound. Relatively high Km values were also calculated for monochloro-substituted cis,cis-muconates (300-500 microM), in contrast with the low Km value of 20 microM for 2,4-dichloro-cis,cis-muconate. The catalytic constant of the pure enzyme was 3820 min-1 when measured with 2,4-dichloro-cis,cis-muconate.

Alcaligenes↗

PubMed1989

Purification and characterization of 4-methylmuconolactone methyl-isomerase, a novel enzyme of the modified 3-oxoadipate pathway in nocardioform actinomycetes.

The novel enzyme 4-methyl-2-enelactone methyl-isomerase was detected in, and purified to electrophoretic homogeneity from, p-toluate-grown cells of Rhodococcus rhodocrous N75, a nocardioform actinomycete. The enzyme was very thermostable and had a native Mr of 75,500; as the monomer had an Mr of 17,000, the enzyme is probably tetrameric. The new isomerase is highly specific with respect to its lactone substrate, only accepting (+)-(4S)-4-methylmuconolactone (4-carboxymethyl-4-methylbut-2-en-1,4-olide), and the putative isomerization reaction intermediate 1-methylbislactone ((-)-1-methyl-3,7-dioxo-2,6-dioxabicyclo-[3.3.0]octane) as substrates, and yielding (-)-(4S)-3-methylmuconolactone (4-carboxymethyl-3-methylbut-2-en-1,4-olide) as product. Some other lactone analogues acted as competitive inhibitors. Our data suggest that the isomerization does not involve actual methyl migration, but proceeds via the 1-methybislactone.

Chromatography, Gel↗

PubMed1987

Assemblage of ortho cleavage route for simultaneous degradation of chloro- and methylaromatics.

Genetic engineering is a powerful means of accelerating the evolution of new biological activities and has considerable potential for constructing microorganisms that can degrade environmental pollutants. Critical enzymes from five different catabolic pathways of three distinct soil bacteria have been combined in patchwork fashion into a functional ortho cleavage route for the degradation of methylphenols and methylbenzoates. The new bacterium thereby evolved was able to degrade and grow on mixtures of chloro- and methylaromatics that were toxic even for the bacteria that could degrade the individual components of the mixtures. Except for one enzymatic step, the pathway was fully regulated and its component enzymes were only synthesized in response to the presence of pathway substrates.

Alcaligenes↗