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

https://mmbr.asm.org/content/mmbr/43/1/73.full.pdf

Chromosomal genetics of Pseudomonas.

Protein phosphatase-2B was purified from extracts of rabbit skeletal muscle by a procedure that involved fractionation with ammonium sulphate, chromatography on DEAE-Sepharose, fractionation with poly(ethylene glycol), gel filtration on Sephadex G-200 (Mr = 98000 +/- 4000), chromatography on Affi-Gel Blue and affinity chromatography on calmodulin-Sepharose. The enzyme was purified 3500-fold in seven days with an overall yield of 0.5%. The alpha-subunit of phosphorylase kinase, protein phosphatase inhibitor-1 and the myosin P-light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase-2B with similar kinetic constants. The alpha-subunit of phosphorylase kinase was dephosphorylated at least 100-fold more rapidly than the beta-subunit, while glycogen phosphorylase, glycogen synthase, histones H1 and H2B, ATP-citrate lyase, acetyl-CoA carboxylase, L-pyruvate kinase and protein synthesis initiation factor eIF-2 were not dephosphorylated at significant rates. Protein phosphatase-2B became activated 10-fold by calmodulin (A0.5 = 6 nM) after chromatography on DEAE-Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the Vmax of the reaction without altering the Km for inhibitor-1. The activity of protein phosphatase-2B was completely dependent on Ca2+ in the presence or absence of calmodulin. Half-maximal activation was observed at 1.0 microM Ca2+ in the absence, and at 0.5 microM Ca2+ in the presence, of 0.03 microM calmodulin. Protein phosphatase-2B was inhibited completely by trifluoperazine; half-maximal inhibition occurred at 45 microM in the absence and 35 microM in the presence of 0.03 microM calmodulin. The metabolic role of protein phosphatase-2B in vivo is discussed in the light of the observation that this enzyme is probably identical to a major calmodulin-binding protein of neural tissue termed calcineurin or CaM-BP80 [Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Lett. 137, 80-84].

The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle.

The catechol and protocatechuate branches of the 3-oxoadipate pathway, which are important for the bacterial degradation of aromatic compounds, converge at the common intermediate 3-oxoadipate enol-lactone. A 3-oxoadipate enol-lactone-hydrolyzing enzyme, purified from benzoate-grown cells of Rhodococcus opacus (erythropolis) 1CP, was found to have a larger molecular mass under denaturing conditions than the corresponding enzymes previously purified from gamma-proteobacteria. Sequencing of the N terminus and of tryptic peptides allowed cloning of the gene coding for the 3-oxoadipate enol-lactone hydrolase by using PCR with degenerate primers. Sequencing showed that the gene belongs to a protocatechuate catabolic gene cluster. Most interestingly, the hydrolase gene, usually termed pcaD, was fused to a second gene, usually termed pcaC, which encodes the enzyme catalyzing the preceding reaction, i.e., 4-carboxymuconolactone decarboxylase. The two enzymatic activities could not be separated chromatographically. At least six genes of protocatechuate catabolism appear to be transcribed in the same direction and in the following order: pcaH and pcaG, coding for the subunits of protocatechuate 3,4-dioxygenase, as shown by N-terminal sequencing of the subunits of the purified protein; a gene termed pcaB due to the homology of its gene product to 3-carboxy-cis,cis-muconate cycloisomerases; pcaL, the fused gene coding for PcaD and PcaC activities; pcaR, presumably coding for a regulator of the IclR-family; and a gene designated pcaF because its product resembles 3-oxoadipyl coenzyme A (3-oxoadipyl-CoA) thiolases. The presumed pcaI, coding for a subunit of succinyl-CoA:3-oxoadipate CoA-transferase, was found to be transcribed divergently from pcaH.

Characterization of a Protocatechuate Catabolic Gene Cluster from Rhodococcus opacus 1CP: Evidence for a Merged Enzyme with 4-Carboxymuconolactone-Decarboxylating and 3-Oxoadipate Enol-Lactone-Hydrolyzing Activity

The bacterial energy-transducing NADH-quinone oxidoreductases.

Evolutionary divergence of co-selected beta-ketoadipate enol-lactone hydrolases in Acinetobacter calcoaceticus.

Repetitions in the NH2-terminal amino acid sequence of beta-ketoadipate enol-lactone hydrolase from Pseudomonas putida.

Homologous amino acid sequences in enzymes mediating sequential metabolic reactions.

gamma-Carboxymuconolactone decarboxylase (EC 4.1.1.44) from Azotobacter vinelandii resembled the isofunctional enzymes from Acinetobacter calcoaceticus and Pseudomonas putida. All three decarboxylases appeared to be hexamers formed by association of identical subunits of about 13,300 daltons. The A. vinelandii and P. putida decarboxylases cross-reacted immunologically with each other, and the NH2-terminal amino acid sequences of the enzymes differed in no more than 7 of the first 36 residues. In contrast, the A. calcoaceticus decarboxylase did not cross-react with the decarboxylase from A. vinelandii or P. putida; the NH2-terminal amino acid sequences of these enzymes diverged about 50% from the NH2-terminal amino acid sequence of the A. calcoaceticus decarboxylase.

L N Ornston

Papers

Evolutionary divergence of co-selected beta-ketoadipate enol-lactone hydrolases in Acinetobacter calcoaceticus.

Repetitions in the NH2-terminal amino acid sequence of beta-ketoadipate enol-lactone hydrolase from Pseudomonas putida.

Homologous amino acid sequences in enzymes mediating sequential metabolic reactions.

Evolutionary relationships among gamma-carboxymuconolactone decarboxylases.