New Crystallographic Snapshots of Large Domain Movements in Bacterial 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) catalyzes the first committed step of the mevalonate pathway, which is used across biology in the biosynthesis of countless metabolites. HMGR consumes 2 equiv of the cofactor NAD(P)H to perform the four-electron reduction of HMG-CoA to mevalonate toward the production of steroids and isoprenoids, the largest class of natural products. Recent structural data have shown that HMGR contains a highly mobile C-terminal domain (CTD) that is believed to adopt many different conformations to permit binding and dissociation of the substrate, cofactors, and products at specific points during the reaction cycle. Here, we have characterized the HMGR from Delftia acidovorans as an NADH-specific enzyme and determined crystal structures of the enzyme in unbound, mevalonate-bound, and NADH- and citrate-bound states. Together, these structures depict ligand binding in both the active site and the cofactor-binding site while illustrating how a conserved helical motif confers NAD(P)H cofactor specificity. Unexpectedly, the NADH-bound structure also reveals a new conformation of the CTD, in which the domain has "flipped" upside-down, while directly binding the cofactor. By capturing these structural snapshots, this work not only expands the known range of HMGR domain movement but also provides valuable insight into the catalytic mechanism of this biologically important enzyme.

Initial O2 Insertion Step of the Tryptophan Dioxygenase Reaction Proposed by a Heme-Modification Study.

L-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme that catalyzes the production of N-formylkynurenine by inserting O2 into the pyrrole ring of L-tryptophan. Although a ferrous-oxy form (Fe²âº-O2) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain elusive. In this study, the O2 insertion reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using a heme-modification approach, which allowed us to draw a quantitative correlation between the inductive electronic effects of the heme substituents and the substituent-induced changes in the functional behaviors of the ferrous-oxy form. We succeeded in preparing reconstituted PaTDO with synthetic hemes, which were different with respect to the inductive electron-withdrawing nature of the heme substituents at positions 2 and 4. An increase in the electron-withdrawing power of the heme substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme substituents, the lower the electron density on the heme iron. The decrease in the electron density of the heme iron resulted in a higher frequency shift of the C-O stretch of the heme-bound CO and enhanced the dissociation of O2 from the ferrous-oxy intermediate. This result was interpreted as being due to weaker π back-donation from the heme iron to the bound CO or O2. More importantly, the reaction rates of the ferrous-oxy intermediate to oxidize L-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous-oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O2 to the indole ring of L-Trp.

In vitro evidence of chain transfer to tetraethylene glycols in enzymatic polymerization of polyhydroxyalkanoate.

A polyhydroxyalkanoate (PHA) was enzymatically synthesized in vitro, and the end structure of PHA associated with a chain transfer (CT) reaction was investigated. In the CT reaction, PHA chain transfers from PHA synthase (PhaC) to a CT agent, resulting in covalent bonding of CT agent to the PHA chain at its carboxyl end. In vitro CT reaction has never been demonstrated because of relatively low yields of in vitro synthesized poly[(R)-3-hydroxybutyrate)] (P(3HB)), which makes it difficult to characterize the end structures of the polymers by nuclear magnetic resonance (NMR). To overcome these difficulties, a novel in vitro synthesis method that produced relatively larger amounts of P(3HB) was developed by employing PhaCDa from Delftia acidovorans and two enantioselective enoyl-coenzyme A (CoA) hydratases which were R-hydratase (PhaJAc) from Aeromonas caviae and S-hydratase (FadB1x) from Pseudomonas putida KT2440 with ß-butyrolactone and CoA as starting materials. Using this method, P(3HB) synthesis was performed with tetraethylene glycols (TEGs) as a discriminable CT agent, and the resultant P(3HB) was characterized by (1)H-NMR. NMR analysis revealed that the carboxylic end of P(3HB) was covalently linked to TEGs, providing the first direct evidence of in vitro CT reaction.

Characterization of polyhydroxyalkanoate (PHA) synthase derived from Delftia acidovorans DS-17 and the influence of PHA production in Escherichia coli.

Heterologous expression of polyhydroxyalkanoate (PHA) synthase from Delftia acidovorans DS-17 (PhaC(Da)) in Escherichia coli JM109 leads to effective production of high-molecular-weight poly[(R)-3-hydroxybutyrate] [P(3HB)]. This study examined the effect of PhaC(Da) expression on P(3HB) production in E. coli JM109 (Da strain) by comparing with the strain expressing PHA synthase (PhaC(Re)) from Ralstonia eutropha (Re strain). First, the kinetic properties of PhaC(Da) were investigated. Among the five detergents examined, Triton X-100 remarkably activated PhaC(Da), as well as PhaC(Re). The affinity of PhaC(Da) for its substrate was lower than that of PhaC(Re), whereas the maximum reaction rate of PhaC(Da) was higher than that of PhaC(Re). However, the kinetic differences were not likely to influence P(3HB) production in the cells. Under conditions of P(3HB) production, the translational levels of monomer-supplying enzymes (PhaA and PhaB) were similar in both the Da and Re strains, whereas PhaC exhibited different expression levels: the abundance of soluble PhaC(Da) was lower than that of soluble PhaC(Re). This observation suggests that the production of high-molecular-weight P(3HB) by the Da strain would be attributed to the low amounts of active PhaC(Da) in the cells.

Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases.

Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD(+)-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.

4-hydroxyphenylpyruvate dioxygenase catalysis: identification of catalytic residues and production of a hydroxylated intermediate shared with a structurally unrelated enzyme.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.

Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes.

Engineered glyphosate resistance is the most widely adopted genetically modified trait in agriculture, gaining widespread acceptance by providing a simple robust weed control system. However, extensive and sustained use of glyphosate as a sole weed control mechanism has led to field selection for glyphosate-resistant weeds and has induced significant population shifts to weeds with inherent tolerance to glyphosate. Additional weed control mechanisms that can complement glyphosate-resistant crops are, therefore, urgently needed. 2,4-dichlorophenoxyacetic acid (2,4-D) is an effective low-cost, broad-spectrum herbicide that controls many of the weeds developing resistance to glyphosate. We investigated the substrate preferences of bacterial aryloxyalkanoate dioxygenase enzymes (AADs) that can effectively degrade 2,4-D and have found that some members of this class can act on other widely used herbicides in addition to their activity on 2,4-D. AAD-1 cleaves the aryloxyphenoxypropionate family of grass-active herbicides, and AAD-12 acts on pyridyloxyacetate auxin herbicides such as triclopyr and fluroxypyr. Maize plants transformed with an AAD-1 gene showed robust crop resistance to aryloxyphenoxypropionate herbicides over four generations and were also not injured by 2,4-D applications at any growth stage. Arabidopsis plants expressing AAD-12 were resistant to 2,4-D as well as triclopyr and fluroxypyr, and transgenic soybean plants expressing AAD-12 maintained field resistance to 2,4-D over five generations. These results show that single AAD transgenes can provide simultaneous resistance to a broad repertoire of agronomically important classes of herbicides, including 2,4-D, with utility in both monocot and dicot crops. These transgenes can help preserve the productivity and environmental benefits of herbicide-resistant crops.

Declining capacity of starving Delftia acidovorans MC1 to degrade phenoxypropionate herbicides correlates with oxidative modification of the initial enzyme.

Bioremediation relies on the stability of enzymatic activities, particularly when bioavailable contaminant concentrations do not permit much renewal of microbial biomass. Starving Delftia acidovorans MC1 were found to lose specific degradation activity, while accumulating variants of the alpha-ketoglutarate-dependent dioxygenase RdpA, the enzyme initiating the degradation of (RS)-2-(2,4-dichlorophenoxy)propionate. These variants differed in their pI and originated from post-translational modification, since there is only one rdpA gene in the genome. It was tested if RdpA modification resulted from carbonylation by reactive oxygen species, known side products of dioxygenase reactions. Carbonylated amino acids in proteins of starved cells were specifically derivatized with 2,4-dinitrophenylhydrazine. Subsequent immunolabeling of the resulting hydrazones and mass spectrometry of tryptic digests confirmed different levels of carbonylation of RdpA.

Chain transfer reaction catalyzed by various polyhydroxyalkanoate synthases with poly(ethylene glycol) as an exogenous chain transfer agent.

Polyhydroxyalkanoate (PHA) synthases catalyze chain transfer (CT) reaction after polymerization reaction of PHA by transferring PHA chain from PHA synthase to a CT agent, resulting in covalent bonding of CT agent to PHA chain at the carboxyl end. Previous studies have shown that poly(ethylene glycol) (PEG) is an effective exogenous CT agent. This study aimed to compare the effects of PEG on CT reaction during poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis by using six PHA synthases in Escherichia coli JM109. The synthesized P(3HB) polymers were characterized in terms of molecular weight and end-group structure. Supplementation of PEG to the culture medium reduced P(3HB) molecular weights by up to 96% due to PEG-induced CT reaction. The P(3HB) polymers were subjected to (1)H NMR analysis to confirm the formation of a covalent bond between PEG and P(3HB) chain at the carboxyl end. This study revealed the reactivity of PHA synthases to PEG with respect to CT reaction in E. coli.

Bacterial sulfite dehydrogenases in organotrophic metabolism: separation and identification in Cupriavidus necator H16 and in Delftia acidovorans SPH-1.

The utilization of organosulfonates as carbon sources by aerobic or nitrate-reducing bacteria usually involves a measurable, uncharacterized sulfite dehydrogenase. This is tacitly assumed to be sulfite : ferricytochrome-c oxidoreductase [EC 1.8.2.1], despite negligible interaction with (eukaryotic) cytochrome c: the enzyme is assayed at high specific activity with ferricyanide as electron acceptor. Purified periplasmic sulfite dehydrogenases (SorAB, SoxCD) are known from chemoautotrophic growth and are termed 'sulfite oxidases' by bioinformatic services. The catalytic unit (SorA, SoxC; termed 'sulfite oxidases' cd02114 and cd02113, respectively) binds a molybdenum-cofactor (Moco), and involves a cytochrome c (SorB, SoxD) as electron acceptor. The genomes of several bacteria that express a sulfite dehydrogenase during heterotrophic growth contain neither sorAB nor soxCD genes; others contain at least four paralogues, for example Cupriavidus necator H16, which is known to express an inducible sulfite dehydrogenase during growth with taurine (2-aminoethanesulfonate). This soluble enzyme was enriched 320-fold in four steps. The 40 kDa protein (denatured) had an N-terminal amino acid sequence which started at position 42 of the deduced sequence of H16_B0860 (termed 'sulfite oxidase' cd02114), which we named SorA. The neighbouring gene is an orthologue of sorB, and the sorAB genes were co-transcribed. Cell fractionation showed SorA to be periplasmic. The corresponding enzyme in Delftia acidovorans SPH-1 was enriched 270-fold, identified as Daci_0055 (termed 'sulfite oxidase' cd02110) and has a cytochrome c encoded downstream. We presume, from genomic data for bacteria and archaea, that there are several subgroups of sulfite dehydrogenases, which all contain a Moco, and transfer electrons to a specific cytochrome c.

N-acetyltaurine dissimilated via taurine by Delftia acidovorans NAT.

The naturally occurring sulfonate N-acetyltaurine was synthesized chemically and its identity was confirmed. Aerobic enrichment cultures for bacteria able to utilize N-acetyltaurine as sole source of fixed nitrogen or as sole source of carbon were successful. One representative isolate, strain NAT, which was identified as a strain of Delftia acidovorans, grew with N-acetyltaurine as carbon source and excreted stoichiometric amounts of sulfate and ammonium. Inducible enzyme activities were measured in crude extracts of this organism to elucidate the degradative pathway. Cleavage of N-acetyltaurine by a highly active amidase yielded acetate and taurine. The latter was oxidatively deaminated by taurine dehydrogenase to ammonium and sulfoacetaldehyde. This key intermediate of sulfonate catabolism was desulfonated by the known reaction of sulfoacetaldehyde acetyltransferase to sulfite and acetyl phosphate, which was further degraded to enter central metabolism. A degradative pathway including transport functions is proposed.

Purification, characterization, gene cloning and nucleotide sequencing of D: -stereospecific amino acid amidase from soil bacterium: Delftia acidovorans.

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40 degrees C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45 degrees C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd2+, Ag+, Zn2+, Hg2+ and As3+ . The NH2 terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.

Localization and characterization of two novel genes encoding stereospecific dioxygenases catalyzing 2(2,4-dichlorophenoxy)propionate cleavage in Delftia acidovorans MC1.

Two novel genes, rdpA and sdpA, encoding the enantiospecific alpha-ketoglutarate dependent dioxygenases catalyzing R,S-dichlorprop cleavage in Delftia acidovorans MC1 were identified. Significant similarities to other known genes were not detected, but their deduced amino acid sequences were similar to those of other alpha-ketoglutarate dioxygenases. RdpA showed 35% identity with TauD of Pseudomonas aeruginosa, and SdpA showed 37% identity with TfdA of Ralstonia eutropha JMP134. The functionally important amino acid sequence motif HX(D/E)X(23-26)(T/S)X(114-183)HX(10-13)R/K, which is highly conserved in group II alpha-ketoglutarate-dependent dioxygenases, was present in both dichlorprop-cleaving enzymes. Transposon mutagenesis of rdpA inactivated R-dichlorprop cleavage, indicating that it was a single-copy gene. Both rdpA and sdpA were located on the plasmid pMC1 that also carries the lower pathway genes. Sequencing of a 25.8-kb fragment showed that the dioxygenase genes were separated by a 13.6-kb region mainly comprising a Tn501-like transposon. Furthermore, two copies of a sequence similar to IS91-like elements were identified. Hybridization studies comparing the wild-type plasmid and that of the mutant unable to cleave dichlorprop showed that rdpA and sdpA were deleted, whereas the lower pathway genes were unaffected, and that deletion may be caused by genetic rearrangements of the IS91-like elements. Two other dichlorprop-degrading bacterial strains, Rhodoferax sp. strain P230 and Sphingobium herbicidovorans MH, were shown to carry rdpA genes of high similarity to rdpA from strain MC1, but sdpA was not detected. This suggested that rdpA gene products are involved in the degradation of R-dichlorprop in these strains.

High-level expression and characterization of a highly functional Comamonas acidovorans xanthine dehydrogenase in Pseudomonas aeruginosa.

An improved procedure is described for the high-level expression of Comamonas acidovorans XDH in Pseudomonas aeruginosa PAO1-LAC. The level of functional expression (56 mg protein/L culture) is found to be 7-fold higher than that observed in Escherichia coli and 30-fold higher than that induced in C. acidovorans. Co-expression of the xdhC gene is required for maximal level of functional expression. Comparison of purified preparations of XDH expressed in the absence of xdhC (XDH(AB)) with that expressed in its presence (XDH(ABC)) shows the increased level of activity due to the level of Mo incorporation. The Fe and FAD contents of expressed enzymes are independent of xdhC co-expression. Electron paramagnetic resonance spectroscopy, circular dichroism spectroscopy, metal analysis, and kinetic properties of recombinant purified XDH(ABC) are identical with those exhibited by the native enzyme. This expression system should serve as a valuable tool for further biophysical and mechanistic investigations of xanthine dehydrogenase by site-directed mutagenesis. A method is also described to evaluate the suitability of P. aeruginosa and other organisms as potential expression hosts for five different sources of xdh genes.

Regulation of catabolic enzymes during long-term exposure of Delftia acidovorans MC1 to chlorophenoxy herbicides.

Delftia acidovorans MC1 is able to grow on chlorophenoxy herbicides such as 2,4-dichlorophenoxypropionic acid (2,4-DCPP) and 2,4-dichlorophenoxyacetic acid as sole sources of carbon and energy. High concentrations of the potentially toxic organics inhibit the productive degradation and poison the organism. To discover the target of chlorophenoxy herbicides in D. acidovorans MC1 and to recognize adaptation mechanisms, the response to chlorophenoxy acids at the level of proteins was analysed. The comparison of protein patterns after chemostatic growth on pyruvate and 2,4-DCPP facilitated the discovery of several proteins induced and repressed due to the substrate shifts. Many of the induced enzymes, for example two chlorocatechol 1,2-dioxygenases, are involved in the metabolism of 2,4-DCPP. A stronger induction of some catabolic enzymes (chlorocatechol 1,2-dioxygenase TfdC(II), chloromuconate cycloisomerase TfdD) caused by an instant increase in the concentration of 2,4-DCPP resulted in increased rates of productive detoxification and finally in resistance of the cells. Nevertheless, the decrease of the (S)-2,4-DCPP-specific 2-oxoglutarate-dependent dioxygenase in 2D gels reveals a potential bottleneck in 2,4-DCPP degradation. Well-known heat-shock proteins and oxidative-stress proteins play a minor role in adaptation, because apart from DnaK only a weak or no induction of the proteins GroEL, AhpC and SodA was observed. Moreover, the modification of elongation factor Tu (TufA), a strong decrease of asparaginase and the induction of the hypothetical periplasmic protein YceI point to additional resistance mechanisms against chlorophenoxy herbicides.

Delftia acidovorans MC1 resists high herbicide concentrations--a study of nutristat growth on (RS)-2-(2,4-Dichlorophenoxy)propionate and 2,4-dichlorophenoxyacetate.

Delftia acidovorans MC1 was continuously cultivated under nutristat conditions with elevated concentrations of the herbicides (RS)-2-(2,4-dichlorophenoxy)propionate [(RS)-2,4-DP] and 2,4-dichlorophenoxyacetate (2,4-D). The presence of 1-5 mM of either of these compounds did not essentially inhibit growth. Moreover, substrate consumption was not essentially affected at pH values of 7.0-9.0 selected by reason of alkaline in situ conditions found e.g. on contaminated building rubble but was decreased at pH 9.3. The adenylate energy charge declined to some degree as the herbicide concentration rose, the extent of this increasing as the pH rose. This was caused by an increase in the concentration of ADP and in particular AMP, in contrast to the fairly constant ATP level of around 4 nmol/mg dry mass with (RS)-2,4-DP and 2 nmol/mg with 2,4-D. Comparison of the individual growth parameters with theoretical data taking into account maintenance coefficients of 0.48 mmol (RS)-2,4-DP/g*h and 0.6 mmol 2,4-D/g*h revealed that the culture followed purely kinetic rules. This excludes the necessity of using substrate to a significant extent to satisfy extra efforts in energy for homeostasic work under these accentuated conditions.

An extra large insertion in the polyhydroxyalkanoate synthase from Delftia acidovorans DS-17: its deletion effects and relation to cellular proteolysis.

The polyhydroxyalkanoate (PHA) synthase (PhaC(Da)) from Delftia acidovorans DS-17 (formerly Comamonas acidovorans) has a unique large insertion consisting of 40 amino acid residues in the alpha/beta hydrolase fold region. In order to examine whether this insertion is necessary for enzyme function, we generated a mutant gene where the nucleotides encoding the insertion sequence were deleted [phaC(Da)del(342-381)]. The ability of the mutant PhaC(Da) lacking the insertion sequence to produce PHA in recombinant Escherichia coli JM109 was compared with that of wild-type PhaC(Da). The results revealed that the mutant enzyme had approximately one fourth the activity of the wild-type enzyme. However, there was no significant difference in PHA content accumulated in cells harboring either the mutant PhaC(Da) or wild-type PhaC(Da) nor were there any differences in the molecular masses of the produced polymers. Therefore, we have concluded that the characteristic insertion is not indispensable for PHA synthesis. Also, slight cellular proteolysis in E. coli was found specifically for wild-type PhaC(Da) by Western blot analysis. This result prompted us to further examine the proteolytic stability of PhaC(Da) in D. acidovorans. Consequently, it has been suggested that the insertion region of PhaC(Da) is susceptible to cellular proteolysis during accumulation of PHA.

Physiological and genetic characteristics of two bacterial strains utilizing phenoxypropionate and phenoxyacetate herbicides.

Two strains, Rhodoferax sp. P230 and Delftia (Comamonas) acidovorans MCI, have previously been shown to carry activities for the degradation of the two enantiomers of (RS)-2-(2,4-dichlorophenoxy-)propionate (dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy-)propionate (mecoprop) and, in addition, are capable of degrading phenoxyacetate derivatives 2.4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA). Metabolism of the herbicides is initiated by alpha-ketoglutarate-dependent dioxygenases for both enantiomers of the phenoxypropionate herbicides and for 2,4-D. These activities were constitutively expressed for both enantiomers of dichlorprop in strain MC1 and for the Renantiomer in strain P230. Enzyme activities for the complete degradation of phenoxyacetate and phenoxypropionate herbicides were induced during incubation on either of these herbicides. Strain MC1 has about threefold higher activities for the degradation of dichlorprop and for growth on this substrate (mumax = 0.15 h(-1)) than strain P230; the maximum growth rate on 2,4-D amounts to 0.045 h(-1) with strain MC1. Dichlorprop is utilized faster than mecoprop and the R-enantiomers are cleaved with higher rates than the S-enantiomers. The degradation of the chlorophenolic intermediates seems to proceed via the modified ortho cleavage pathway as indicated by activities of the respective enzymes. The enzymatic results were supported by genetic investigations by which the presence of the genes tfdB (encoding a dichlorophenol hydroxylase), tfdC (encoding a chlorocatechol 1,2-dioxygenase) and tfdD (encoding a chloromuconate cycloisomerase) could be demonstrated in both strains by PCR after application of respective primers. The presence of the tfdA gene (encoding a 2,4-D/alpha-ketoglutarate dioxygenase) was only shown for strain P230 but was lacking in strain MC1. Sequence analysis of the tfd gene fragments revealed high homology to the degradative genes of other proteobacterial strains degrading chloroaromatic compounds. Strain MC1 carries a plasmid of about 120 kb which apparently harbors herbicide degradative genes as concluded from deletion mutants which have lost 2,4-D[phenoxalkanoate]/alpha-ketoglutarate dioxygenase activities for cleavage of the R- and S-enantiomer, and of 2,4-D. For strain P230, no plasmid could be demonstrated; the activity was stably conserved in this strain during growth under nonselective conditions.

[Studies on the metabolism pathway of aniline degradation by Comamonas acidovorans AN3].

All of the enzymes relating to aniline degradation in C. acidovorans AN3 were inducible ones. When growing on aniline as sole carbon, nitrogen and energy sources, the cells of C. acidovorans AN3 contained aniline dioxygenase, catechol-2, 3-dioxygenase, 2-hydromuconic semialdehyde dehydrogenase, 4-oxalocrotonic acid decarboxylase and 4-hydroxy-2-oxovalerate acid aldolase. The Km and Vmax of aniline dioxygenase were 292 mumol/L and 3.57 mumol.mg-1. min-1, respectively. The Km and Vmax of catechol-2, 3-dioxygenase were 16.4 mumol/L and 15.2 mumol.mg-1.min-1, respectively. According to these results, the degradation metabolism pathway of aniline by C. acidovorans AN3 was hypothesised.