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Biomedical subjects

W K Yeh

Publications and source records attributed to W K Yeh.

At least 37 records · Page 2Linked to original sources
PubMed1985

Purification and properties of ferredoxinTOL. A component of toluene dioxygenase from Pseudomonas putida F1.

Toluene dioxygenase oxidizes toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. This reaction is catalyzed by a multienzyme system that is induced in cells of Pseudomonas putida F1 during growth on toluene. One of the components of toluene dioxygenase has been purified to homogeneity and shown to be an iron-sulfur protein that has been designated ferredoxinTOL. The molecular weight of ferredoxinTOL was calculated to be 15,300, and the purified protein was shown to contain 2 g of atoms each of iron- and acid-labile sulfur which appear to be organized as a single [2Fe-2S]cluster. Solutions of ferredoxinTOL were brown in color and showed absorption maxima at 277, 327, and 460 nm. A shoulder in the spectrum of the oxidized protein was discernible at 575 nm. Reduction with sodium dithionite or NADH and ferredoxinTOL reductase resulted in a decrease in visible absorbance at 460 and 575 nm, with a concomitant shift in absorption maxima to 382 and 438 nm. The redox potential of ferredoxinTOL was estimated to be -109 mV. In the oxidized state, the protein is diamagnetic. However, upon reduction it exhibited prominent electron paramagnetic resonance signals with anisotropy in g values (gx = 1.81, gy = 1.86, and gz = 2.01). Anaerobic reductive titrations revealed that ferredoxinTOL is a one-electron carrier that accepts electrons from NADH in a reaction that is mediated by a flavoprotein (ferredoxinTOL reductase). The latter is the first component in the toluene dioxygenase system. Reduced ferredoxinTOL can transfer electrons to cytochrome c or to a terminal iron-sulfur dioxygenase (ISP-TOL) which catalyzes the incorporation of molecular oxygen into toluene and related aromatic substrates.

Amino Acids↗
PubMed1984

High-performance liquid chromatographic assay for S-adenosyl-L-methionine: macrocin O-methyltransferase.

A high-performance liquid chromatographic (HPLC) procedure was developed to assay S-adenosyl-L-methionine: macrocin O-methyltransferase. This enzyme catalyzes the rate-limiting terminal reaction of tylosin biosynthesis in Streptomyces fradiae. HPLC analysis was improved by resin treatment of cell-free extracts to remove endogenous tylosin and related compounds. Relomycin was selected as an internal standard and the enzymatic reaction conditions were optimized. The reaction mixture was extracted with ethyl acetate to recover the substrate, product and the internal standard. Efficient separation of the macrolide antibiotics was provided by ion-pair reversed-phase HPLC. An average relomycin recovery was 90%. The O-methyltransferase activity could be routinely and reproducibly determined by monitoring tylosin formation at 285 nm.

Cations, Divalent↗
PubMed1984

p-Chloromercuribenzoate specifically modifies thiols associated with the active sites of beta-ketoadipate enol-lactone hydrolase and succinyl CoA: beta-ketoadipate CoA transferase.

beta-Ketoadipate enol-lactone hydrolase (EC 3.1.1.24) and succinyl CoA: beta-ketoadipate transferase (EC 2.8.3.6) catalyze consecutive metabolic reactions in bacteria. The enzymes appear to be members of different families of related proteins. Enzymes within the enol-lactone hydrolase family appear to have diverged so extensively that common ancestry sometimes is not directly evident from comparison of NH2-terminal amino acid sequences of the proteins. Amino acid sequences at or near the active sites of the enzymes are likely to have been conserved, and hence a chemical proble that reacted specifically near the active sites of the enzymes might identify regions of amino acid sequence in which evolutionary affinities among widely divergent proteins could be identified. p-Chloromercuribenzoate appears to be such a probe because enol-lactone hydrolases and CoA transferases from Acinetobacter calcoaceticus and Pseudomonas putida were completely inhibited by stoichiometric quantities of the compound which appears to modify selectively cysteinyl side chains at or near the active sites of the enzymes. Stoichiometric inhibition of P. putida enol-lactone hydrolase was observed in the presence of excess dithiothreitol; therefore the reactive cysteinyl residue in this enzyme appears to be nucleophilic. The hydrolase is inhibited by beta-ketoadipate, but the compound must be supplied at 10 mM concentrations in order to achieve 50% inhibition, so the product inhibition is unlikely to be significant under physiological conditions.

Acinetobacter↗
PubMed1982

Overlapping evolutionary affinities revealed by comparison of amino acid compositions.

Comparison of the amino acid compositions of purified proteins indicates the presence of overlapping evolutionary affinities among enzymes of the beta-ketoadipate pathway. Isofunctional enzymes from different bacterial genera share a common evolutionary origin. Moreover, enzymes that mediate isofunctional or chemically analogous reactions within an organism appear to be evolutionarily homologous. Most remarkably, closely similar amino acid compositions are found in enzymes that mediate the following consecutive metabolic steps: lactonization, decarboxylation, hydrolysis, and transfer of a thioester bond.

Adipates↗
PubMed1982

Similar structures in gamma-carboxymuconolactone decarboxylase and beta-ketoadipate succinyl coenzyme A transferase.

gamma-Carboxymuconolactone decarboxylase (EC 4.1.1.44) and beta-ketoadipate succinyl coenzyme A transferase (EC 2.8.3.6) mediate different steps in the beta-ketoadipate pathway. Antisera prepared against the Pseudomonas putida transferase cross-reacted immunologically with the decarboxylase from the same organism. The transferase is formed by association of two nonidentical protein subunits. The NH2-terminal amino acid sequences of the two nonidentical transferase subunits resembled each other and also were similar to the NH2-terminal amino acid sequence of the decarboxylase.

Acinetobacter↗
PubMed1981

Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida.

Cells of Pseudomonas putida, after growth with toluene, contain a multicomponent enzyme system that oxidizes toluene to (+)-1(S),2(R)-dihydroxy-3-methyl-cyclohexa-3,5-diene. One of these components has been purified to homogeneity and shown to be a flavoprotein that contains FAD as the only detectable prosthetic group. Fad was removed from the enzyme during purification. However, equilibrium dialysis experiments showed that the enzyme can bind one mol of FAD/mol of enzyme protein. The apparent molecular weight of the enzyme is 46,000, as judged by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. The latter result suggests the presence of a single polypeptide chain. The amino acid composition of the enzyme reveals a relatively high content of the hydrophobic amino acids leucine, isoleucine, and valine and is remarkably similar in composition to the flavoproteins that function in certain monooxygenase enzyme systems. The purified enzyme catalyzes the reduction of dichloroindophenol, nitrobluetetrazolium, ferricyanide, and ferredoxinTOL. Its ability to reduce cytochrome c and to function in the toluene dioxygenase enzyme system is absolutely dependent on the presence of ferredoxinTOL.

Amino Acids↗
PubMed1981

Evolutionarily homologous alpha 2 beta 2 oligomeric structures in beta-ketoadipate succinyl-CoA transferases from Acinetobacter calcoaceticus and Pseudomonas putida.

Homogeneous beta-ketoadipate succinyl-CoA transferase (EC 2.8.3.6) preparations were obtained from extracts of Acinetobacter calcoaceticus and Pseudomonas putida. Gel filtration indicated that the respective transferases have similar molecular weights of 108,000 and 109,000; each transferase appears to have an alpha 2 beta 2 oligomeric structure formed by association of nonidentical subunits with a molecular weight of about 25,000. The subunits were separated by sodium dodecyl sulfate-gel electrophoresis, and differences in their primary structures were revealed by determination of the NH2-terminal amino acid sequences of the oligomers. The transferases cross-react immunologically and possess similar amino acid compositions. These are remarkably similar to the amino acid compositions of gamma-carboxymuconolactone decarboxylases (EC 4.1.1.44) and beta-ketoadipate enol-lactone hydrolases (EC 3.1.1.24), enzymes that mediate consecutive reactions preceding the transferase step in the beta-ketoadipate pathway.

Acinetobacter↗
PubMed1981

Evolutionary relationships among gamma-carboxymuconolactone decarboxylases.

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.

Acinetobacter↗
PubMed1980

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

Muconolactone delta-isomerase (EC 5.3.3.4) and beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24) mediate consecutive reactions in the beta-ketoadipate pathway of bacteria. An earlier investigation (Yeh, W.K., Davis, G., Fletcher, P., and Ornston, L.N. (1978) J. Biol. Chem. 253, 4920-4923) revealed that the respective NH2-terminal amino acid sequences of Pseudomonas putida muconolactone isomerase and Acinetobacter calcoaceticus beta-ketoadipate enol-lactone hydrolase II are evolutionarily homologous. In this report, we describe the purification of Pseudomonas beta-ketoadipate enol-lactone hydrolase and present evidence indicating that the protein is a trimer composed of identical 11,000-dalton subunits. The NH2-terminal amino acid sequences of Pseudomonas muconolactone isomerase and Pseudomonas enol-lactone hydrolase have diverged widely from each other, yet the two sequences contain different fragments of an ancestral sequence which is represented in Acinetobacter enol-lactone hydrolase II. The widely divergent Pseudomonas muconolactone isomerase and Pseudomonas enol-lactone hydrolase sequences each contain unique sets of repeated peptides. In principle, the repetitive sequences might have been introduced by elongation mutations which occurred early in the evolution of the proteins. However, the divergence of Pseudomonas muconolactone isomerase and Pseudomonas enol-lactone hydrolase is so extreme that the observed sequence repetitions cannot have been conserved from ancestral duplication mutations. Rather, the data favor the interpretation that copies of DNA were substituted into structural genes for the enzymes as they diverged.

Adipates↗
PubMed1980

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

Muconolactone isomerase (EC 5.3.3.4) and beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24) mediate consecutive catabolic steps in bacteria. Separately inducible beta-ketoadipate enol-lactone hydrolases I and II are formed in representatives of Acinetobacter calcoaceticus. When subjected to DEAE-cellulose chromatography, Acinetobacter enol-lactone hydrolase I displays heterogeneous behavior which, in whole or in part, appears to be due to modifications of sulfhydryl groups in the protein; the enzyme is unusual in that its NH2-terminal amino acid is cysteine. Comparison of the NH2-terminal amino acid sequence of Acinetobacter enollactone hydrolase I, reported here, with the corresponding amino acid sequences of Acinetobacter enollactone hydrolase II and Pseudomonas enol-lactone hydrolase indicates that all three proteins have diverged widely from a common evolutionary origin. Sequence comparisons suggest that divergence of the Acinetobacter enol-lactone hydrolase structural genes was achieved by substitution with DNA derived from an ancestral muconolactone isomerase structural gene.

Acinetobacter↗
PubMed1980

Homologies in the NH2-terminal amino acid sequences of gamma-carboxymuconolactone decarboxylases and muconolactone isomerases.

gamma-Carboxymuconolactone decarobxylase (EC 4.1.1.44) and muconolactone isomerase (EC 5.3.3.4) mediate chemically analogous reactions in bacteria. The enzymes are inducible, and different metabolites trigger the respective syntheses of the decarboxylases in Acinetobacter calcoaceticus and Pseudomonas putida. The decarobxylases share similar oligomeric structures in which identical subunits of about 13,300 daltons appear to be self-associated into hexamers. Identical residues are found in 18 of the first 36 positions of the enzymes' NH2-terminal amino acid sequences. Thus, genetic rearrangements appear to have placed homologous structural genes for the decarboxylases under different transcriptional control in the two bacterial species. The NH2-terminal amino acid sequences of the decarboxylases and muconolactone isomerases are similar, suggesting that a common ancestral protein gave rise to the enzymes with different (albeit analogous) activities. In addition, the NH2-terminal amino acid sequences of the decarboxylases appear to have been conserved at a second region within the primary structure of the muconolactone isomerases. As has been observed with the two enol-lactone hydrolases (EC 3.1.3.24) of Acinetobacter, the structural genes for the decarboxylases and the isomerases appear to have diverged widely as they were co-selected within a single cell line, In part the divergence appears to have been achieved by mutations in which fragments of DNA within structural genes are replaced with fragments of DNA derived from a co-evolving sequence.

Acinetobacter↗
PubMed1980

Origins of metabolic diversity: substitution of homologous sequences into genes for enzymes with different catalytic activities.

Similar amino acid sequences were found in portions of bacterial enzymes that mediate different biochemical transformations. Reaction catalyzed by the enzymes include oxygenation, decarboxylation, isomerization, and hydrolysis. The proteins share a common evolutionary history because they participate in an overall catabolic process known as the beta-ketoadipate pathway. One interpretation of the sequence similarities might be that duplication of a single gene gave rise to ancestral genes for the enzymes with different catalytic activities. According to this view, homologous sequences from the ancestral gene were conserved as the proteins diverged to assume different functions. This hypothesis is vitiated by comparison of the NH2-terminal amino acid sequences of sets of enzymes that mediate identical or analogous metabolic reactions within an organism. Gene duplications giving rise to the enzymes within each set must have followed duplication of a putative ancestral gene for all the sets. Yet the amino acid sequences of the proteins within each set have diverged widely, and against this background of divergence the conservation of sequences from an ancestor common to all the enzymes is unlikely. Rather, it appears that most regions of sequence similarity shared by enzymes from different sets were acquired subsequent to their divergence from any common ancestor. In some cases it appears that relatively short regions of sequence homology were achieved by mutations causing the transfer of sequence information from one set of structural genes to structural genes in another set. Alignment of homologous amino acid sequences within any single set requires the introduction of few gaps. Because gaps are required to align sequences that have been altered by the insertion of genetic material, the evidence indicates that copies of oligonucleotides were exchanged by genetic substitution among different structural genes as they coevolved.

Acinetobacter↗
PubMed1979

Origins of metabolic diversity: evolutionary divergence by sequence repetition.

Recurring patterns of primary structure have been observed in enzymes that mediate sequential metabolic reactions in bacteria. The enzymes, muconolactone Delta-isomerase [(+)-4-hydroxy-4-carboxymethylisocrotonolactone Delta(2)-Delta(3)-isomerase, EC 5.3.3.4] and beta-ketoadipate enol-lactone hydrolase [4-carboxymethylbut-3-enolide(1,4)enol-lactone-hydrolase, EC 3.1.1.24], have been coselected in bacterial populations because the isomerase can confer no nutritional advantage in the absence of the hydrolase. Similar amino acid sequences recur within the structure of the isomerase, and the amino-terminal amino acid sequence of the isomerase from Pseudomonas putida appears to be evolutionarily homologous with the corresponding sequence of a beta-ketoadipate enol-lactone hydrolase from Acinetobacter calcoaceticus. One interpretation of the sequence repetitions is that they reflect tandem duplication mutations that took place early in the evolution of the proteins. According to this view, the mutations caused elongation of structural genes and the creation of duplicated genes as the metabolic pathways evolved. A review of the sequence data calls attention to a different hypothesis: repeated amino acid sequences were introduced in the course of the proteins' evolution by substitution of copies of DNA sequences into structural genes. Our observations are interpreted on the basis of a model proposing genetic exchange between misaligned DNA sequences. The model predicts that misalignments in one chromosomal region can influence the nature of mutations in another region. Thus, as often has been observed, the mutability of a base pair will be determined by its location in a DNA sequence. Furthermore, the intrachromosomal recombination of DNA sequences may account for complex genetic modifications that occur as new pathways evolve. The model provides an interpretation of an apparent paradox, the rapid creation of new metabolic traits by bacterial genomes that are remarkably resistant to genetic drift.

Amino Acid Sequence↗