The beta-ketoadipate pathway is a chromosomally encoded convergent pathway for aromatic compound degradation that is widely distributed in soil bacteria and fungi. One branch converts protocatechuate, derived from phenolic compounds including p-cresol, 4-hydroxybenzoate and numerous lignin monomers, to beta-ketoadipate. The other branch converts catechol, generated from various aromatic hydrocarbons, amino aromatics, and lignin monomers, also to beta-ketoadipate. Two additional steps accomplish the conversion of beta-ketoadipate to tricarboxylic acid cycle intermediates. Enzyme studies and amino acid sequence data indicate that the pathway is highly conserved in diverse bacteria, including Pseudomonas putida, Acinetobacter calcoaceticus, Agrobacterium tumefaciens, Rhodococcus erythropolis, and many others. The catechol branch of the beta-ketoadipate pathway appears to be the evolutionary precursor for portions of the plasmid-borne ortho-pathways for chlorocatechol degradation. However, accumulating evidence points to an independent and convergent evolutionary origin for the eukaryotic beta-ketoadipate pathway. In the face of enzyme conservation, the beta-ketoadipate pathway exhibits many permutations in different bacterial groups with respect to enzyme distribution (isozymes, points of branch convergence), regulation (inducing metabolites, regulatory proteins), and gene organization. Diversity is also evident in the behavioral responses of different bacteria to beta-ketoadipate pathway-associated aromatic compounds. The presence and versatility of transport systems encoded by beta-ketoadipate pathway regulons is just beginning to be explored in various microbial groups. It appears that in the course of evolution, natural selection has caused the beta-ketoadipate pathway to assume a characteristic set of features or identity in different bacteria. Presumably such identities have been shaped to optimally serve the diverse lifestyles of bacteria.

THE β-KETOADIPATE PATHWAY AND THE BIOLOGY OF SELF-IDENTITY

Aromatic compounds of both natural and man-made sources abound in the environment. The degradation of such chemicals is mainly accomplished by microorganisms. This review provides key background information but centres on recent developments in the bacterial degradation of selected man-made aromatic compounds. An aromatic compound can only be considered to be biodegraded if the ring undergoes cleavage, and this is taken as the major criteria for inclusion in this review (although the exact nature of the enzymic ring-cleavage has not been confirmed in all cases discussed).

The biodegradation of aromatic hydrocarbons by bacteria

https://pure.rug.nl/ws/files/3623614/2006JBiotechnolvBerkel.pdf

Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts.

A review of the occurrence, analyses, toxicity, and biodegradation of naphthenic acids.

Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications.

1
Cyclohexanone oxygenases from Nocardia globerula CL1 and Acinetobacter NCIB 9571 have been purified 12-fold and 35-fold respectively and each gives a single symmetrical sedimentation peak in the ultracentrifuge and a single protein band on 2.25 nm average pore radius polyacrylamide gels.

2
The enzyme from N. globerula has a molecular weight of 53000 while that from Acinetobacter has a molecular weight of about 59000. Each is a single polypeptide chain with one mole of bound FAD per mole of protein that does not dissociate during purification. Acidification of the Acinetobacter enzyme in the presence of (NH4)2SO4 releases the bound FAD and yields native apoenzyme from which the active holoenzyme can be reconstituted. The apparent dissociation constant for the FAD is 40 nM.

3
The near unitary stoichiometry of cyclohexanone, NADPH and oxygen consumption is typical of mixed function oxygenases with external electron donors. The oxygenated product has been identified as 1-oxa-2-oxocycloheptane thus placing these enzymes in the small group of lactone and ester-forming oxygenases. Their correct systematic name is cyclohexanone. NADPH: oxygen oxidoreductase (1,2-lactonizing) (EC 1.14.13.-).

4
A functionally essential sulfhydryl group is present at the catalytic centre of both enzymes but there is no reliable indication from inhibitor studies that they contain any functional metal ion. The three titratable sulfhydryl groups of the Acinetobacter enzyme are not equivalent since reaction with one of them selectively inhibits catalytic activity. Protection against sulfhydryl active agents is afforded by NADPH but not by cyclohexanone.

5
The N. globerula enzyme has a pH optimum of 8.4, apparent Km values of 1.56 μM and 31.3 μM for cyclohexanone and NADPH respectively and a catalytic centre activity of 1018 ml substrate transformed × mol enzyme−1× min−1. The Acinetobacter enzyme has a pH optimum of 9.0, apparent Km values of 6.9 tM and 17.8 μM and a catalytic centre activity of 1390 mol × mol enzyme−1× min−1. Both enzymes display absolute specificity for electron donor which contrasts with the broad specificity for ketone substrate.

6
An enzyme-cyclohexanone complex has been detected by difference spectroscopy only in the case of the Nocardia enzyme. Rapid reduction of the enzyme-bound FAD occurs upon addition of NADPH in the absence of cyclohexanone. Titration of enzyme with NADPH under anaerobic conditions and anaerobic photoreduction in the presence of EDTA have not revealed the formation of any stable flavin semiquinones.

7
These enzymes bear a strong resemblance to several of the monooxygenases that hydroxylate aromatic compounds.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10220.x

The purification and properties of cyclohexanone oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871.

Acinetobacter NCIB 9871 was isolated by elective culture on cyclohexanol and grows with this compound as sole source of carbon It displays a restricted growth spectrum, being unable to grow on a wide range of alternative alicyclic alcohols and ketones Cyclohexanol-grown cells oxidize the growth substrate at a rate of 230 mul of O2/h per mg dry wt with the consumption of 565 mumol of O2/mumol substrate Cyclohexanone is oxidized at a similar rate with the consumption of 485 mumol of O2/mumol 1-Oxa-2-oxocycloheptane and 6-hydroxyhexanoate are both oxidized at the same slow rate of 44 mul of O2/h per mg dry wt and adipate is not oxidized Studies with cell extracts reveal the presence of inducible dehydrogenases for cyclohexanol, 6-hydroxyhexanoate and 6-oxohexanoate and a monooxygenase, that in conjunction with a lactonase converts cyclohexanone to 6-hydroxyhexanoate The monooxygenase is therefore presumed to be of the lactone-forming type and the pathway for conversion of cyclohexanol to adipate; cyclohexanol leads to cyclohexanone leads to 1-oxa-2-oxocycloheptane leads to 6-hydroxyhexanoate leads to 6-oxohexanoate leads to adipate; for which key intermediates have been identified chromatographically, is identical with the route for the oxidation of cyclohexanol by Nocardia globerula CL1

The metabolism of cyclohexanol by Acinetobacter NCIB 9871.

Four Gram-positive bacteria have been isolated from separate soil samples by enrichment culture with acetone as sole source of carbon. Whole cells of all strains grown on acetone rapidly oxidized acetone, acetol and methylglyoxal, and three of the four also oxidized isopropanol. The patterns of induced enzymes in cell extracts are compatible with the oxidation sequence: isopropanol → acetone → acetol → methylglyoxal → pyruvate. Although an enzyme system capable of converting acetone into acetol has not been detected, the inclusion of acetol in the pathway is supported by the results of studies with whole cells and [14C]acetone. The proposed pathway of acetone metabolism is contrasted with evidence for an alternative, but not fully understood, pathway used by Mycobacterium vaccae JOB5.

The Microbial Metabolism of Acetone

1
Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis.

2
The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three or four subunits of molecular weight 54000–58000.

3
The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein.

4
The unitary stoichiometry of cyclopentanone, oxygen and NADPH is typical of mixedfunction oxygenases with external electron donors. The oxygenated product has been identified as 1-oxa-2-oxocyclohexane (5-valerolactone) and enzyme is therefore systematically named as cyclopentanone, NADPH: oxygen oxidoreductase (1,2-lactonizing) (EC 1.14.13.–).

5
Catalytically functional sulfhydryl groups are probably present but the biphasic nature of the inhibition curve when enzyme is titrated with p-hydroxymercuribenzoate reveals the presence of six titratable groups that are not all equivalent.

6
The enzyme has a pH optimum of 7.7. The Km for cyclopentanone is below 1 μM but not experimentally determinable. The response to variation of NADPH concentration is sigmoidal, indicative of homotrophic binding, a Hill plot gave an interaction number of 3, perhaps indicative of cooperativity between three subunits.

7
Anaerobic reduction of enzyme-bound FAD by addition of NADPH required only slightly in excess of two electron equivalents. No evidence for the formation of stable semiquinones was obtained in this or in photoreduction experiments.

8
Though the enzyme displays sensitivity towards transition metal chelating agents the small amounts of Fe and Cu present make a role in electron transport unlikely. The possibility that they may be involved in maintenance of quaternary structure has not been investigated.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10222.x

Purification and Properties of Cyclopentanone Oxygenase of Pseudomonas NCIB 9872

1. Nocardia globerula CL1, isolated by enrichment on cyclohexanol and grown with it as carbon source, oxidized it with a Qo2 of 39μl/h per mg dry wt. and the overall consumption of 2.2μmol of oxygen/mol of substrate. Cyclohexanone, 2-hydroxycyclohexan-1-one dimer and cyclohexane-1,2-dione were oxidized with Qo2 values similar to that for cyclohexanol whereas ∈-caprolactone and 6-hydroxycaproate were oxidized very slowly and adipate not all. 2. Disrupted cell suspensions could not be shown to catalyse the conversion of cyclohexanol into cyclohexanone. 3. A cyclohexanol-induced cyclohexanone oxygenase (specific activity 0.55μmol of NADPH oxidized/min per mg of protein) catalysed the consumption of 1mol of NADPH and 1mol of O2 in the presence of 1mol of cyclohexanone. NADPH oxidation did not occur under anaerobic conditions. The only detected reaction product with 25000g supernatant was 6-hydroxycaproate. 4. Extracts of cyclohexanol-grown cells contained a lactone hydrolase (specific activity 15.6μmol hydrolysed/min per mg of protein), which converted ∈-caprolactone into 6-hydroxycaproate. 5. Incubation of 6-hydroxycaproate with 25000g supernatant in the presence of NAD+ resulted in NAD+ reduction under anaerobic conditions, oxygen consumption under aerobic conditions and the conversion of 6-hydroxycaproate into adipate. 6. Cyclohexanone oxygenase fractions devoid of ∈-caprolactone hydrolase catalysed the stoicheiometric formation of ∈-caprolactone from cyclohexanone in the presence of excess of NADPH. 7. The reaction sequence for the oxidation of cyclohexanone by N. globerula CL1 is: cyclohexanol → cyclohexanone → ∈-caprolactone → 6-hydroxycaproate → adipate. 8. It is suggested that the adipate may be further dissimilated by β-oxidation.

Peter W. Trudgill

Papers

The purification and properties of cyclohexanone oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871.

The metabolism of cyclohexanol by Acinetobacter NCIB 9871.

The Microbial Metabolism of Acetone

Purification and Properties of Cyclopentanone Oxygenase of Pseudomonas NCIB 9872

The metabolism of cyclohexanol by Nocardia globerula CL1.