Properties and functions of two succinic-semialdehyde dehydrogenases from Pseudomonas putida

Two forms of succinic-semialdehyde dehydrogenase have been isolated in Pseudomonas putida. The two enzymes could be separated by filtration on Sephacryl S-300 and their apparent molecular weights were approx. 200,000 and 100,000. The smaller enzyme, which is induced by growth on 4-hydroxyphenylacetate, has been purified to 88% homogeneity by anion-exchange and affinity chromatography. Electrophoresis in sodium dodecyl sulphate gave rise to a molecular weight of 53,000, indicating that the native enzyme is dimeric. Under standard assay conditions this enzyme acts preferentially with NAD but reduces NADP at 9% of the rate observed for NAD. The large enzyme, which is dependent on NADP, is induced by growth on putrescine and its induction is highly coordinated with putrescine: 2-oxoglutarate transaminase, gamma-amino-butyraldehyde dehydrogenase and gamma-aminobutyrate: 2-oxoglutarate transaminase activities. Activity and stability conditions and true Km values for substrate and cosubstrates of the two enzymes were determined.     

Mutations affecting lipoamide dehydrogenases of Pseudomonas putida.

Pseudomonas putida grown on valine produces two lipoamide dehydrogenases, LPD-glu (Mr, 56,000 and LPD-val (Mr, 49,000). The 49,000-dalton protein is used by P. putida for branched-chain keto acid dehydrogenase, whereas the 56,000-dalton protein is presumably used for pyruvate and 2-ketoglutarate dehydrogenases. The objective of this study was to isolate and characterize mutants of P. putida with mutations affecting lipoamide dehydrogenases in order to study the relationship of these two proteins. Mutant JS287 lacked LPD-val, the lipoamide dehydrogenase which is induced by growth on valine and is specific for branched-chain keto acid dehydrogenase, and had normal amounts of LPD-glu, the lipoamide dehydrogenase which is formed during growth on glucose and which is probably used by both pyruvate and 2-ketoglutarate dehydrogenases. Mutant JS94 was a pleiotropic mutant with defects in 2-ketoglutarate, branched-chain, and lipoamide dehydrogenases. Proteolysis of LPD-glu and LPD-val produced completely different digestion products, suggesting that these two proteins are products of separate structural genes. Antisera prepared against LPD-glu reacted only with LPD-glu, whereas antisera prepared against LPD-val reacted with LPD-val and cross-reacted with LPD-glu. Although mutant JS94 did not produce active lipoamide dehydrogenase, cell-free extracts of this mutant contained a protein which cross-reacted with anti-LPD-val.     

Relationship of lipoamide dehydrogenases from Pseudomonas putida to other FAD-linked dehydrogenases

Pseudomonas putida produces two lipoamide dehydrogenases, LPD-glc and LPD-val. LPD-val is specifically required as the lipoamide dehydrogenase of branched-chain keto acid dehydrogenase and LPD-glc fulfills all other requirements for lipoamide dehydrogenase. Both proteins are dimers with one FAD per subunit. LPD-glc has an absorption maximum at 455 nm, but LPD-val has a maximum at 460 nm. Comparison of amino acid compositions revealed that LPD-glc was more closely related to Escherichia coli and pig heart lipoamide dehydrogenase than to LPD-val. LPD-val did not appear to be closely related to any of the proteins compared with the possible exception of mercuric reductase.     

Comparison of two dioxygenases from Pseudomonas putida.

Catechol 2,3-dioxygenase and homoprotocatechuate 2,3-dioxygenase were purified from the same strain of Pseudomonas putida. Molecular weights and subunit sizes were similar, but amino acid compositions showed some marked differences.     

Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition.

The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with [185W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of 185W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Fu1 was catalyzed by two different dehydrogenases.     

Purification and characterization of two aldehyde dehydrogenases from Pseudomonas aeruginosa.

Two soluble aldehyde dehydrogenases isoenzymes have been purified and separated from extracts of a paraffin-assimilating bacterium, Pseudomonas aeruginosa. The first one, obtained at an estimated purity of 20% (spec. act. with butanal 0.33 kat/kg) was NAD-dependent. It was rapidly inactivated at pH 8.6 but was efficiently protected by NAD. It had a molecular weight of 225000 and presented a high affinity for aldehydes of short and middle chain lengths. The second enzyme, obtained in a nearly homogenous state (spec. act. with pentanal 0.62 kat/kg) was NADP-dependent. It was activated by ions, in particular potassium ions, and had a good affinity for aldehydes of higher chain lengths. Both enzymes were stabilized by thiols and glycerol and were inactivated by reagents of sulfhydryl groups. These enzymes are 'constitutive' and their physiological function is uncertain. When the bacteria were grown on n-paraffin a new membrane-bound NAD-dependent aldehyde dehydrogenase activity was produced.     

Three distinct quinoprotein alcohol dehydrogenases are expressed when Pseudomonas putida is grown on different alcohols.

A bacterial strain that can utilize several kinds of alcohols as its sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida HK5. Three distinct dye-linked alcohol dehydrogenases (ADHs), each of which contained the prosthetic group pyrroloquinoline quinone (PQQ), were formed in the soluble fractions of this strain grown on different alcohols. ADH I was formed most abundantly in the cells grown on ethanol and was similar to the quinoprotein ADH reported for P. putida (H. Görisch and M. Rupp, Antonie Leeuwenhoek 56:35-45, 1989) except for its isoelectric point. The other two ADHs, ADH IIB and ADH IIG, were formed separately in the cells grown on 1-butanol and 1,2-propanediol, respectively. Both of these enzymes contained heme c in addition to PQQ and functioned as quinohemoprotein dehydrogenases. Potassium ferricyanide was an available electron acceptor for ADHs IIB and IIG but not for ADH I. The molecular weights were estimated to be 69,000 for ADH IIB and 72,000 for ADH IIG, and both enzymes were shown to be monomers. Antibodies raised against each of the purified ADHs could distinguish the ADHs from one another. Immunoblot analysis showed that ADH I was detected in cells grown on each alcohol tested, but ethanol was the most effective inducer. ADH IIB was formed in the cells grown on alcohols of medium chain length and also on 1,3-butanediol. Induction of ADH IIG was restricted to 1,2-propanediol or glycerol, of which the former alcohol was more effective. These results from immunoblot analysis correlated well with the substrate specificities of the respective enzymes. Thus, three distinct quinoprotein ADHs were shown to be synthesized by a single bacterium under different growth conditions.     

Chromium reduction in Pseudomonas putida.

Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a Km of 40 microM CrO4(2-). Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.     

Pseudomonas putida KT2440 responds specifically to chlorophenoxy herbicides and their initial metabolites

Pseudomonas putida KT2440 is often used as a model to investigate toxicity mechanisms and adaptation to hazardous chemicals in bacteria. The objective of this paper was to test the impact of the chlorophenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-(2,4-dichlorophenoxy)propanoic acid (DCPP) and their metabolites 2,4-dichlorophenol (DCP) and 3,5-dichlorocatechol (DCC), on protein expression patterns and physiological parameters. Both approaches showed that DCC has a different mode of action and induces different responses than DCPP, 2,4-D and DCP. DCC was the most toxic compound and was active as an uncoupler of oxidative phosphorylation. It repressed the synthesis of ferric uptake regulator (Fur)-dependent proteins, e.g. fumarase C and L-ornithine N5-oxygenase, which are involved in oxidative stress response and iron uptake. DCPP, 2,4-D and DCP were less toxic than DCC. They disturbed oxidative phosphorylation to a lesser extent by a yet unknown mechanism. Furthermore, they repressed enzymes of energy-consuming biosynthetic pathways and induced membrane transporters for organic substrates. A TolC homologue component of multidrug resistance transporters was found to be induced, which is probably involved in the removal of lipophilic compounds from membranes.     

Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1

UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD(+)-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The K(m) values of PA2022 and PA3559 for UDP-glucose are approximately 0.1 and 0.4 mM, whereas the K(m) values for NAD(+) are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg(2+), which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.     

Myo-inositol transport system in Pseudomonas putida.

The kinetic features of the myo-inositol transport system in Pseudomonas putida are reported. The system is sensitive to osmotic shock, is not operative in membrane vesicles, and does not involved substrate phosphorylation. Line-weaver-Burk plots indicate the presence of two different systems, whose Kt are 5 micrometer and 0.43 mM and whose V max are 7.9 and 27 nml/mg per min, respectively. Transport activity of glucose-grown cells is very low. myo-Inositol-grown cells lose the high-affinity system upon osmotic shock; concentrated shock fluid possesses myo-inositol-binding activity. The system is very specific for the myo-configuration of the cyclitol.     

Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida

The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.     

The aromatic alcohol dehydrogenases in Pseudomonas putida N.C.I.B. 9869 grown on 3,5-xylenol and p-cresol.

Whole cells of Pseudomonas putida N.C.I.B 9869, when grown on either 3,5-xylenol or p-cresol, oxidized both m- and p-hydroxybenzyl alcohols. Two distinct NAD+-dependent m-hydroxybenzyl alcohol dehydrogenases were purified from cells grown on 3,5-xylenol. Each is active with a range of aromatic alcohols, including both m- and p-hydroxybenzyl alcohol, but differ in their relative rates with the various substrates. An NAD+-dependent alcohol dehydrogenase was also partially purified from p-cresol grown cells. This too was active with m- and p-hydroxybenzyl alcohol and other aromatic alcohols, but was not identical with either of the other two dehydrogenases. All three enzymes were unstable, but were stabilized by dithiothreitol and all were inhibited with p-chloromercuribenzoate. All were specific for NAD+ and each was shown to catalyse conversion of alcohol into aldehyde.     

alpha-Pinene metabolism by Pseudomonas putida.

By using metabolically altered mutants and acrylate, novel putative intermediates of alpha-pinene metabolism by Pseudomonas putida PIN11 were detected. They were characterized as 3-isopropylbut-3-enoic acid and (zeta)-2-methyl-5-isopropylhexa-2,5-dienoic acid.     

Peptide utilization by Pseudomonas putida and Pseudomonas maltophilia.

Pseudomonas putida assimilates peptides and hydrolyses them with intracellular peptidases. Amino acid auxotrophs (his, trp, thr or met) grew on a variety of di- and tripeptides up to twice as slowly as with free amino acids. Pseudomonas putida has separate uptake systems for both dipeptides and oligopeptides (three or more residues). Although the dipeptide system transported a variety of structurally diverse dipeptides it did not transport peptides having either unprotonatable N-terminal amino groups, blocked C-terminal carboxyl groups, D-residues, three or more residues, N-methylated peptide bonds, or beta-amino acids. Oligopeptide uptake lacked amino acid side-chain specificity, required a free N-terminal L-residue and had an upper size limit. Glycylglycyl-D,L-p-fluorophenylalanine inhibited growth of P. putida. Uptake of glycylglycyl[I-14C]alanine was rapid and inhibited by 2,4-dinitrophenol. Both dipeptide and oligopeptide uptake were constitutive. Dipeptides competed with oligopeptides for oligopeptide uptake, but oligopeptides did not compete in the dipeptide system. Final bacterial yields were 5 to 10 times greater when P. putida his was grown on histidyl di- or tripeptides rather than on free histidine because the histidyl residue was protected from catabolism by L-histidine ammonia-lyase. Methionine peptides could satisfy the methionine requirements of P. maltophilia. Generation times on glycylmethionine and glycylmethionylglycine were equal to those obtained with free methionine. Methionylglycylmethionylmethionine gave a generation time twice that of free methionine. Growth of P. maltophilia was inhibited by glycylglycyl-D,L-p-fluorophenylalanine.     

Regulation of alkane oxidation in Pseudomonas putida.

We have studied the appearance of whole-cell oxidizing activity for n-alkanes and their oxidation products in strains of Pseudomonas putida carrying the OCT plasmid. Our results indicate that the OCT plasmid codes for inducible alkane-hydroxylating and primary alcohol-dehydrogenating activities and that the chromosome codes for constitutive oxidizing activities for primary alcohols, aliphatic aldehydes, and fatty acids. Mutant isolation confirms the presence of an alcohol dehydrogenase locus on the OCT plasmid and indicated the presence of multiple alcohol and aldehyde dehydrogenase loci on the P. putida chromosome. Induction tests with various compounds indicate that inducer recognition has specificity for chain length and can be affected by the degree of oxidation of the carbon chain. Some inducers are neither growth nor respiration substrates. Growth tests with and without a gratuitous inducer indicate that undecane is not a growth substrate because it does not induce alkane hydroxylase activity. Using a growth test for determining induction of the plasmid alcohol dehydrogenase it is possible to show that heptane induces this activity in hydroxylase-negative mutants. This suggests that unoxidized alkane molecules are the physiological inducers of both plasmid activities.