Publications

• Complete nucleotide sequence and functional analysis of the genes for 2-aminophenol metabolism from Pseudomonas sp. AP-3.

  Takenaka S., Murakami S., Kim Y.J., Aoki K.

  A 13.9-kb region, which contained the 2-aminophenol 1,6-dioxygenase genes (amnBA) reported before, was cloned from the 2-aminophenol-assimilating bacterium Pseudomonas sp. AP-3. The complete nucleotide sequence of this region was determined and six genes were found downstream of amnBA. The eight g ... >> More

  A 13.9-kb region, which contained the 2-aminophenol 1,6-dioxygenase genes (amnBA) reported before, was cloned from the 2-aminophenol-assimilating bacterium Pseudomonas sp. AP-3. The complete nucleotide sequence of this region was determined and six genes were found downstream of amnBA. The eight genes together were designated amnBACFEDHG. Each gene was similar to the corresponding gene operating in the meta-cleavage pathway, except for amnB, amnA, and amnD. The four 2-aminophenol-metabolizing enzymes, 2-aminomuconic 6-semialdehyde dehydrogenase, 2-aminomuconate deaminase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase, were purified and characterized. NH2-terminal amino acid sequences of each purified enzyme agreed with those deduced from amnC, amnF, amnE, and amnD, respectively. These genes were therefore assigned as the genes encoding these respective proteins. The tight clustering of the amn genes, which were all transcribed in the same direction, raised the possibility that these genes formed a single operon. The organization of the amn genes was entirely different from that of the atd, dmp, and xyl genes reported in the meta-cleavage pathway, although these latter genes clustered similarly. << Less

  Arch. Microbiol. 174:265-272(2000) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600.

  Shingler V., Marklund U., Powlowski J.

  The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pa ... >> More

  The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pathway. The genes for the entire pathway were previously found to be clustered, and the nucleotide sequences of dmpKLMNOPBC and D, which encode the first four biochemical steps of the pathway, were determined. By using a combination of deletion mapping, nucleotide sequence determinations, and polypeptide analysis, we identified the remaining six genes of the pathway. The fifteen genes, encoded in the order dmpKLMNOPQBCDEFGHI, lie in a single operon structure with intergenic spacing that varies between 0 to 70 nucleotides. Homologies found between the newly determined gene sequences and known genes are reported. Enzyme activity assays of deletion derivatives of the operon expressed in Escherichia coli were used to correlate dmpE, G, H, and I with known meta-cleavage enzymes. Although the function of the dmpQ gene product remains unknown, dmpF was found to encode acetaldehyde dehydrogenase (acylating) activity (acetaldehyde:NAD+ oxidoreductase [coenzyme A acylating]; E.C.1.2.1.10). The role of this previously unknown meta-cleavage pathway enzyme is discussed. << Less

  J. Bacteriol. 174:711-724(1992) [PubMed] [EuropePMC]

• Kinetic and stereochemical analysis of YwhB, a 4-oxalocrotonate tautomerase homologue in Bacillus subtilis: mechanistic implications for the YwhB- and 4-oxalocrotonate tautomerase-catalyzed reactions.

  Wang S.C., Johnson W.H. Jr., Czerwinski R.M., Stamps S.L., Whitman C.P.

  YwhB, a 4-oxalocrotonate tautomerase (4-OT) homologue in Bacillus subtilis, has no known biological role, and the gene has no apparent genomic context. The kinetic and stereochemical properties of YwhB have been examined using available enol and dienol compounds. The kinetic analysis shows that Yw ... >> More

  YwhB, a 4-oxalocrotonate tautomerase (4-OT) homologue in Bacillus subtilis, has no known biological role, and the gene has no apparent genomic context. The kinetic and stereochemical properties of YwhB have been examined using available enol and dienol compounds. The kinetic analysis shows that YwhB has a relatively nonspecific 1,3- and 1,5-keto-enol tautomerase activity, with the former activity prevailing. Replacement of Pro-1 or Arg-11 with an alanine significantly reduces or abolishes these activities, implicating both residues as critical ones for the activities. In D2O, ketonization of two monoacid substrates (2-hydroxy-2,4-pentadienoate and phenylenolpyruvate) produces a mixture of stereoisomers {2-keto-3-[2H]-4-pentenoate and 3-[2H]-phenylpyruvate}, where the (3R)-isomers predominate. Ketonization of 2-hydroxy-2,4-hexadienedioate, a diacid, in D2O affords mostly the opposite enantiomer, (3S)-2-oxo-[3-2H]-4-hexenedioate. The mono- and diacids apparently bind in different orientations in the active site of YwhB, but the highly stereoselective nature of the YwhB reaction using a diacid suggests that the biological substrate for YwhB may be a diacid. Moreover, of the three dienols examined, 1,3- and 1,5-keto-enol tautomerization reactions are only observed for 2-hydroxy-2,4-hexadienedioate, indicating that the C-3 and C-5 positions are accessible for protonation in this compound. Incubation of 4-OT with 2-hydroxy-2,4-hexadienedioate in D2O results in a racemic mixture of 2-oxo-[3-2H]-4-hexenedioate, suggesting that 4-OT may not catalyze a 1,3-keto-enol tautomerization reaction using this dienol. It has previously been shown that 4-OT catalyzes the near stereospecific conversion of 2-hydroxy-2,4-hexadienedioate to (5S)-[5-2H]-2-oxo-3-hexenedioate in D2O. Taken together, these observations suggest that 4-OT might function as a 1,5-keto-enol tautomerase using 2-hydroxy-2,4-hexadienedioate. << Less

  Biochemistry 46:11919-11929(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Expression and stereochemical and isotope effect studies of active 4-oxalocrotonate decarboxylase.

  Stanley T.M., Johnson W.H. Jr., Burks E.A., Whitman C.P., Hwang C.C., Cook P.F.

  4-Oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH) from Pseudomonas putida mt-2 form a complex that converts 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate in the catechol meta fission pathway. To facilitate mechanistic and structural studies of the complex, the two enzymes ... >> More

  4-Oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH) from Pseudomonas putida mt-2 form a complex that converts 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate in the catechol meta fission pathway. To facilitate mechanistic and structural studies of the complex, the two enzymes have been coexpressed and the complex has been purified to homogeneity. In addition, Glu-106, a potential catalytic residue in VPH, has been changed to glutamine, and the resulting E106QVPH mutant has been coexpressed with 4-OD and purified to homogeneity. The 4-OD/E106QVPH complex retains full decarboxylase activity, with comparable kinetic parameters to those observed for 4-OD in the wild-type complex, but is devoid of any detectable hydratase activity. Decarboxylation of (5S)-2-oxo-3-[5-D]hexenedioate by either the 4-OD/VPH complex or the mutant complex generates 2-hydroxy-2,4E-[5-D]pentadienoate in D(2)O. Ketonization of 2-hydroxy-2,4-pentadienoate by the wild-type complex is highly stereoselective and results in the formation of 2-oxo-(3S)-[3-D]-4-pentenoate, while the mutant complex generates a racemic mixture. These results indicate that 2-hydroxy-2, 4-pentadienoate is the product of 4-OD and that 2-oxo-4-pentenoate results from a VPH-catalyzed process. On this basis, the previously proposed hypothesis for the conversion of 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate has been revised [Lian, H., and Whitman, C. P. (1994) J. Am. Chem. Soc. 116, 10403-10411]. Finally, the observed (13)C kinetic isotope effect on the decarboxylation of 2-oxo-3-hexenedioate by the 4-OD/VPH complex suggests that the decarboxylation step is nearly rate-limiting. Because the value is not sensitive to either magnesium or manganese, it is likely that the transition state for carbon-carbon bond cleavage is late and that the metal positions the substrate and polarizes the carbonyl group, analogous to its role in oxalacetate decarboxylase. << Less

  Biochemistry 39:718-726(2000) [PubMed] [EuropePMC]

• Uncovering the protocatechuate 2,3-cleavage pathway genes.

  Kasai D., Fujinami T., Abe T., Mase K., Katayama Y., Fukuda M., Masai E.

  Paenibacillus sp. (formerly Bacillus macerans) strain JJ-1b is able to grow on 4-hydroxybenzoate (4HB) as a sole source of carbon and energy and is known to degrade 4HB via the protocatechuate (PCA) 2,3-cleavage pathway. However, none of the genes involved in this pathway have been identified. In ... >> More

  Paenibacillus sp. (formerly Bacillus macerans) strain JJ-1b is able to grow on 4-hydroxybenzoate (4HB) as a sole source of carbon and energy and is known to degrade 4HB via the protocatechuate (PCA) 2,3-cleavage pathway. However, none of the genes involved in this pathway have been identified. In this study, we identified and characterized the JJ-1b genes for the 4HB catabolic pathway via the PCA 2,3-cleavage pathway, which consisted of praR and praABEGFDCHI. Based on the enzyme activities of cell extracts of Escherichia coli carrying praI, praA, praH, praB, praC, and praD, these genes were found to code for 4HB 3-hydroxylase, PCA 2,3-dioxygenase, 5-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, 2-hydroxymuconate-6-semialdehyde dehydrogenase, 4-oxalocrotonate (OCA) tautomerase, and OCA decarboxylase, respectively, which are involved in the conversion of 4HB into 2-hydroxypenta-2,4-dienoate (HPD). The praE, praF, and praG gene products exhibited 45 to 61% amino acid sequence identity to the corresponding enzymes responsible for the catabolism of HPD to pyruvate and acetyl coenzyme A. The deduced amino acid sequence of praR showed similarity with those of IclR-type transcriptional regulators. Reverse transcription-PCR analysis revealed that praABEGFDCHI constitute an operon, and these genes were expressed during the growth of JJ-1b on 4HB and PCA. praR-praABEGFDCHI conferred the ability to grow on 4HB to E. coli, suggesting that praEGF were functional for the conversion of HPD to pyruvate and acetyl coenzyme A. A promoter analysis suggested that praR encodes a repressor of the pra operon. << Less

  J. Bacteriol. 191:6758-6768(2009) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Metabolism of 2-aminophenol by Pseudomonas sp. AP-3: modified meta-cleavage pathway.

  Takenaka S., Murakami S., Shinke R., Aoki K.

  A novel pathway for 2-aminophenol metabolism by Pseudomonas sp. AP-3 is proposed. The proposed pathway is similar to that known for meta-cleavage of catechol except that one of the hydroxyl groups on the metabolites is replaced by an amino group. During the degradation of 2-aminophenol, 2-amino-2, ... >> More

  A novel pathway for 2-aminophenol metabolism by Pseudomonas sp. AP-3 is proposed. The proposed pathway is similar to that known for meta-cleavage of catechol except that one of the hydroxyl groups on the metabolites is replaced by an amino group. During the degradation of 2-aminophenol, 2-amino-2,4-pentadienoic acid is the last metabolite containing an amino group. We, therefore, propose a modified meta-cleavage pathway for the 2-aminophenol metabolism. << Less

  Arch Microbiol 170:132-137(1998) [PubMed] [EuropePMC]