Purification and Characterization of Two Enantioselective α-Ketoglutarate-Dependent Dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH

α-Ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and α-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His₆-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the α-ketoglutarate-dependent dioxygenases and share the specific motif HXDX₂₄TX₁₃₁HX₁₀R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent K_m values were 99 μM for (R)-mecoprop, 164 μM for (R)-dichlorprop, and 3 μM for α-ketoglutarate for RdpA and 132 μM for (S)-mecoprop, 495 μM for (S)-dichlorprop, and 20 μM for α-ketoglutarate for SdpA. Both enzymes had high apparent K_m values for oxygen; these values were 159 μM for SdpA and >230 μM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace α-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAα-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.     

Separation of two dichlorprop/α-ketoglutarate dioxygenases with enantiospecific properties from Comamonas acidovorans MC1

Comamonas acidovorans MC1, which is capable of degrading the chiral phenoxypropionate herbicides 2-(2,4-dichlorophenoxy)propionate [dichlorprop, (RS)-2,4-DP] and 2-(4-chloro-2-methylphenoxy)propionate [mecoprop, (RS)-MCPP] and of degrading the phenoxyacetate herbicides 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), was investigated with respect to the enzymatic basis of this broad substrate specificity. The initial steps of the degradation pathway of (RS)-2,4-DP and 2,4-D were studied. By applying either ion exchange chromatography or hydrophobic interaction chromatography it was possible to separate two enzyme fractions with etherolytic activity, which exhibited pronounced substrate specificity. One enzyme fraction was highly specific for the degradation of the R-enantiomer of 2,4-DP and did not essentially attack the S-configuration. The other enzyme fraction showed pronounced activity toward the cleavage of the S-enantiomer and additionally utilized 2,4-D with almost equal velocity; (R)-2,4-DP was even cleaved at a low rate by this enzyme. These results confirm the existence of phenoxyalkanoatedegrading enzymes with enantiospecific properties in strain MC1.     

Stereospecific degradation of the phenoxypropionate herbicide dichlorprop

2,4-Dichlorophenoxyacetic acid (2,4-D)/α-ketoglutarate (α-KG) dioxygenase, TfdA, from Ralstonia eutropha JMP134, was purified from recombinant cells and shown by gas chromatographic and colorimetric methods to degrade only the S enantiomer of dichlorprop, a phenoxypropionate herbicide. Similarly, cell extracts of Burkholderia cepacia RASC, containing a biochemically and genetically related α-KG-dependent dioxygenase, also were shown to oxidize (S)-dichlorprop using chiral HPLC and colorimetric methods. In contrast, cell extracts of a mecoprop-degrading strain of Alcaligenes denitrificans were shown to catabolize (R)-dichlorprop. Although the A. denitrificans activity exhibited stereospecificity opposite to that of the JMP134 and RASC strains, its cofactor requirements were found to be characteristic of an α-KG-dependent dioxygenase. A PCR amplification product from the DNA of this strain was shown to encode an amino acid sequence that was 95% and 86% identical to the corresponding region of TfdA in RASC and JMP134, respectively. Thus, closely related herbicide-degrading gene products appear to be capable of exhibiting opposite stereochemical degradative capabilities.     

Structural basis for the enantiospecificities of R- and S-specific phenoxypropionate/alpha-ketoglutarate dioxygenases

(R)- and (S)-dichlorprop/alpha-ketoglutarate dioxygenases (RdpA and SdpA) catalyze the oxidative cleavage of 2-(2,4-dichlorophenoxy)propanoic acid (dichlorprop) and 2-(4-chloro-2-methyl-phenoxy)propanoic acid (mecoprop) to form pyruvate plus the corresponding phenol concurrent with the conversion of alpha-ketoglutarate (alphaKG) to succinate plus CO2. RdpA and SdpA are strictly enantiospecific, converting only the (R) or the (S) enantiomer, respectively. Homology models were generated for both enzymes on the basis of the structure of the related enzyme TauD (PDB code 1OS7). Docking was used to predict the orientation of the appropriate mecoprop enantiomer in each protein, and the predictions were tested by characterizing the activities of site-directed variants of the enzymes. Mutant proteins that changed at residues predicted to interact with (R)- or (S)-mecoprop exhibited significantly reduced activity, often accompanied by increased Km values, consistent with roles for these residues in substrate binding. Four of the designed SdpA variants were (slightly) active with (R)-mecoprop. The results of the kinetic investigations are consistent with the identification of key interactions in the structural models and demonstrate that enantiospecificity is coordinated by the interactions of a number of residues in RdpA and SdpA. Most significantly, residues Phe171 in RdpA and Glu69 in SdpA apparently act by hindering the binding of the wrong enantiomer more than the correct one, as judged by the observed decreases in Km when these side chains are replaced by Ala.     

Fate of the herbicides mecoprop, dichlorprop, and 2,4-D in aerobic and anaerobic sewage sludge as determined by laboratory batch studies and enantiomer-specific analysis

Aerobic degradation experiments with the racemic mixtures of mecoprop and dichlorprop revealed that activated sludge collected from the aeration tank of a municipal waste water treatment plant degraded both enantiomers of mecoprop and dichlorprop within 7 days, albeit in an enantioselective manner; the (S) enantiomers were preferentially degraded. Mecoprop, dichlorprop, and 2,4-D were completely metabolized under aerobic conditions, as shown by the 86–98% elimination of dissolved organic carbon. Under anaerobic conditions, the concentration of 2,4-D decreased exponentially with a first-order reaction rate constant of 0.24 per day and without a lag-phase. After an incubation time of 17 days, 2,4-D was completely removed. 2,4-Dichlorophenol was the main metabolite of anaerobic 2,4-D degradation; only traces of 4-chlorophenol were detected. In contrast, the chiral phenoxypropionic acid herbicides mecoprop and dichlorprop persisted under anaerobic conditions during 49 days of incubation.     

Synthesis of Optically Active 1,4-Thiazane-3-carboxylic Acid via Optical Resolution by Preferential Crystallization of (RS)-2-Amino-3-[(2-chloroethyl)sulfanyl]propanoic Acid Hydrochloride

Optically active 1,4-thiazane-3-carboxylic acid [TCA] was synthesized from cysteine via optical resolution by preferential crystallization. The intermediate (RS)-2-amino-3-[(2-chloroethyl)sulfanyl]propanoic acid hydrochlo-ride [(RS)-ACS?HCl] was found to exist as a conglomerate based on its melting point, solubility and IR spectrum. (RS)-ACS?HCl was optically resolved by preferential crystallization to yield (R)- and (S)-ACS?HCl. (R)- and (S)-ACS?HCl thus obtained were recrystallized from a mixture of hydrochloric acid and 2-propanol, taking account of the solubility of (RS)-ACS?HCl, efficiently yielding both enantiomers in optically pure forms. (R)- and (S)-TCA were then respectively synthesized by the cyclization of (R)- and (S)-ACS?HCl in ethanol in the presence of triethylamine.     

Biodegradation of mixtures of chlorophenoxyalkanoic acid herbicides by Alcaligenes denitrificans

An Alcaligenes denitrificans strain able to degrade (R)-2-(2-methyl-4-chlorophenoxy)propionic acid [(R)-MCPP, mecoprop] was assessed for its ability to utilise a range of chlorophenoxyalkanoic acid herbicides in single, binary, tertiary and quaternary combinations in batch culture. Degradation rates were rapid with single growth substrates; complete degradation occurred within 29 h for 2,4-dichlorophenoxyacetic acid (2,4-D), 43 h for 4-chloro-2-methylphenoxyacetic acid (MCPA) and 50 h for (R)-MCPP, respectively. After 20 h, the degradation of (RS)-2-(2,4-dichlorophenoxy)propionic acid [(RS)-2,4-DP] had ceased, with only the (R)-enantiomer being degraded. In binary combination, 2,4-D and MCPP degraded within 55 h. Degradation rates decreased when herbicides were added in tertiary and quaternary combinations. Thus, at the whole cell level, catalysis of closely related herbicides is likely to be facilitated by diverse enzymatic activity in A. denitrificans. Journal of Industrial Microbiology & Biotechnology (2000) 25, 255–259. Received 16 April 2000/ Accepted in revised form 07 August 2000     

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 α-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 α-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_114-183HX_10-13R/K, which is highly conserved in group II α-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.     

Purification and characterization of two enantioselective alpha-ketoglutarate-dependent dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH

Alpha-ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and alpha-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His6-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the alpha-ketoglutarate-dependent dioxygenases and share the specific motif HXDX(24)TX(131)HX(10)R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent Km values were 99 microM for (R)-mecoprop, 164 microM for (R)-dichlorprop, and 3 microM for alpha-ketoglutarate for RdpA and 132 microM for (S)-mecoprop, 495 microM for (S)-dichlorprop, and 20 microM for alpha-ketoglutarate for SdpA. Both enzymes had high apparent Km values for oxygen; these values were 159 microM for SdpA and >230 microM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace alpha-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAalpha-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.     

Genetic Analysis of Phenoxyalkanoic Acid Degradation in Sphingomonas herbicidovorans MH

Phenoxyalkanoic acid degradation is well studied in Beta- and Gammaproteobacteria, but the genetic background has not been elucidated so far in Alphaproteobacteria. We report the isolation of several genes involved in dichlor- and mecoprop degradation from the alphaproteobacterium Sphingomonas herbicidovorans MH and propose that the degradation proceeds analogously to that previously reported for 2,4-dichlorophenoxyacetic acid (2,4-D). Two genes for α-ketoglutarate-dependent dioxygenases, sdpA_MH and rdpA_MH, were found, both of which were adjacent to sequences with potential insertion elements. Furthermore, a gene for a dichlorophenol hydroxylase (tfdB), a putative regulatory gene (cadR), two genes for dichlorocatechol 1,2-dioxygenases (dccA_I/II), two for dienelactone hydrolases (dccD_I/II), part of a gene for maleylacetate reductase (dccE), and one gene for a potential phenoxyalkanoic acid permease were isolated. In contrast to other 2,4-D degraders, the sdp, rdp, and dcc genes were scattered over the genome and their expression was not tightly regulated. No coherent pattern was derived on the possible origin of the sdp, rdp, and dcc pathway genes. rdpA_MH was 99% identical to rdpA_MC1, an (R)-dichlorprop/α-ketoglutarate dioxygenase from Delftia acidovorans MC1, which is evidence for a recent gene exchange between Alpha- and Betaproteobacteria. Conversely, DccA_I and DccA_II did not group within the known chlorocatechol 1,2-dioxygenases, but formed a separate branch in clustering analysis. This suggests a different reservoir and reduced transfer for the genes of the modified ortho-cleavage pathway in Alphaproteobacteria compared with the ones in Beta- and Gammaproteobacteria.     

Relating repair susceptibility of carcinogen-damaged DNA with structural distortion and thermodynamic stability

A key issue in the nucleotide excision repair (NER) of bulky carcinogen–DNA adducts is the ability of the NER machinery to recognize and repair certain adducts while failing to repair others. Unrepaired adducts can survive to cause mutations that initiate the carcinogenic process. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region polycyclic aromatic hydrocarbon, can be metabolically activated to the enantiomeric benzo[c]phenanthrene diol epoxides (B[c]PhDEs), (+)-(1S,2R,3R,4S)-3,4- dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phe nanthrene and the corresponding (–)-(1R,2S,3S,4R) isomer. These react predominantly with adenine residues in DNA to produce the stereoisomeric 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts. Duplexes containing the 1R (+) or 1S (–) B[c]Ph-dA adduct in codon 61 of the human N-ras mutational hotspot sequence CA*A, with B[c]Ph modification at A*, are not repaired by the human NER system. However, the analogous stereoisomeric DNA adducts of the bay region benzo[a]pyrene diol epoxide (B[a]PDE), 10S (+)- and 10R (–)-trans-anti-B[a]P-N⁶-dA, are repaired in the same base sequence. In order to elucidate structural and thermodynamic origins of this phenomenon, we have carried out a 2 ns molecular dynamics simulation for the 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts in an 11mer duplex containing the human N-ras codon 61 sequence, and compared these results with our previous study of the B[a]P-dA adducts in the same sequence. The molecular mechanics Poisson– Boltzmann surface area (MM-PBSA) method was applied to calculate the free energies of the pair of stereoisomeric B[c]Ph-dA adducts, and a detailed structural analysis was carried out. The different repair susceptibilities of the B[a]P-dA adducts and the B[c]Ph-dA adducts can be attributed to different degrees of distortion, stemming from combined effects of differences in the quality of Watson–Crick hydrogen bonding, unwinding, stretching and helix backbone perturbations. These differences are due to the different intrinsic topologies of the rigid, planar bay region adducts versus the twisted, sterically hindered fjord region adducts.     

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg⁻¹ h⁻¹, which is the highest ever reported for a plant system.     

Sphingomonas herbicidovorans MH: a versatile phenoxyalkanoic acid herbicide degrader

Sphingomonas herbicidovorans MH was isolated from a dichlorprop-degrading soil column. It is able to grow on phenoxyalkanoic acid herbicides, such as mecoprop, dichlorprop, 2,4-D, MCPA, and 2,4-DB. Strain MH utilizes both enantiomers of the chiral herbicides mecoprop and dichlorprop as sole carbon and energy sources. Enantiomer-specific uptake systems are responsible for transporting the acidic substrates across the cell membrane. Catabolism is initiated by two enantiomer-specific α-ketoglutarate-dependent dioxygenases that catalyze the cleavage of the ether bond of the respective enantiomer to yield the corresponding phenol and pyruvate. Therefore selective degradation of the enantiomers of mecoprop and dichlorprop by strain MH is not only due to enantioselective catabolism but also to enantioselective transport. Received 07 May 1999/ Accepted in revised form 11 August 1999     

A rapid method to screen degradation ability in chlorophenoxyalkanoic acid herbicide-degrading bacteria

AIMS: An agar medium containing a range of related chlorophenoxyalkanoic acid herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop and racemic 2,4-DP (2-(2,4-dichlorophenoxy) propionic acid) was developed to assess the catabolic activity of a range of degradative strains. METHODS AND RESULTS: The medium was previously developed containing 2,4-D as a carbon source to visualise degradation by the production of dark violet bacterial colonies. Strains isolated on mecoprop were able to degrade 2,4-D, MCPA, racemic mecoprop, (R)-mecoprop and racemic 2,4-DP, whereas the 2,4-D-enriched strains were limited to 2,4-D and MCPA as carbon sources. Sphingomonas sp. TFD44 solely degraded the dichlorinated compounds, 2,4-D, racemic 2,4-DP and 2,4-DB (2,4-dichlorophenoxybutyric acid). However, Sphingomonas sp. AW5, originally isolated on 2,4,5-T, was the only strain to degrade the phenoxybutyric compound MCPB (4-chloro-2-methylphenoxybutyric acid). CONCLUSION: This medium has proved to be a very effective and rapid method for screening herbicide degradation by bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This method reduces the problem of assessing the biodegradability of this family of compounds to an achievable level.     

Optical resolution by preferential crystallization of (RS)-2-amino-3-chloropropanoic acid hydrochloride

The racemic structures of (RS)-2-amino-3-chloropropanoic acid [(RS)-ACP] and (RS)-2-amino-3-chloropropanoic acid hydrochloride [(RS-ACP·HCl] were examined to obtain (R)- and (S)-ACP via optical resolution by preferential crystallization. The melting point, infrared spectrum, solubility, and ternary solubility diagram suggested that (RS)-ACP·HCl exists as a conglomerate and that (RS)-ACP forms a racemic compound. Optical resolution by preferential crystallization of (RS)-ACP·HCl was successfully achieved to yield (R)- and (S)-ACP·HCl. Optically pure (R)- and (S)-ACP were obtained from the purified (R)-and (S)-ACP·HCl, respectively. © 1996 Wiley-Liss, Inc.     

Optical Resolution by Preferential Crystallization of (RS)-2-Amino-3-(2-carboxyethylthio)propanoic Acid

Electrophilic additions of DL- and L-Cys to propenoic acid afforded (RS)- and (R)-2-amino-3-(2-carboxyethylthio)propanoic acids [(RS)- and (R)-ACE], respectively. (RS)-ACE was found to exist as a conglomerate based on its melting point, solubility, and infrared spectrum. (RS)-ACE was optically resolved by preferential crystallization to yield (R)- and (S)-ACE. The obtained (R)- and (S)-ACE were efficiently recrystallized from water, taking account of the solubility of (RS)-ACE, to give them in optically pure form.     

An enzymatic approach to the synthesis of optically pure (3R)- and (3S)-enantiomers of green tea flavor compound 3-hydroxy-3-methylnonane-2,4-dione

Both (3R)- and (3S)-enantiomers of the chiral green tea flavor compound 3-hydroxy-3-methylnonane-2,4-dione were synthesized by the combined use of acetylacetoin synthase and acetylacetoin reductase from Bacillus licheniformis. The first enzyme was utilized to catalyze the homo-coupling of 2,3-octanedione and obtain the enantioenriched (3R)-3-hydroxy-3-methylnonane-2,4-dione (ee 44%). The NADH-dependent acetylacetoin reductase was then employed for the diastereoselective (de > 95%) C2 carbonyl reduction of the sole (3R)-enantiomer of the above 2,4-dione, thus affording the syn diol (2S,3R)-2,3-dihydroxy-3-methylnonan-4-one in enantiomerically pure form. While this step allowed for the recovery of unreacted, optically pure (3S)-3-hydroxy-3-methylnonae-2,4-dione, the corresponding (3R)-enantiomer was obtained by subsequent TEMPO-mediated oxidation of the syn diol intermediate. Moreover, using the title compounds as analytical standards, predominance of the (3R) enantiomer in the natural flavor compound was finally demonstrated by chiral GC–MS analysis.     

Influence of 1-[(E)-2-(2-Methyl-4-Nitrophenyl)Diaz-1-enyl]Pyrrolidine-2-Carboxylic Acid and Diphenyliodonium Chloride on Ruminal Protein Metabolism and Ruminal Microorganisms

The effects of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxylic acid (LY29) and diphenyliodonium chloride (DIC) on the degradation of protein to ammonia were determined in a mixed rumen microbial population taken from sheep on a grass hay-concentrate diet. Both compounds decreased NH₃ production by inhibiting deamination of amino acids. LY29, but not DIC, inhibited growth of the high-activity ammonia-producing species, Clostridium aminophilum and Clostridium sticklandii.     

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Growth of Delftia acidovorans MC1 on 2,4-dichlorophenoxyacetic acid (2,4-D) and on racemic 2-(2,4-dichlorophenoxy)propanoic acid ((RS)-2,4-DP) was studied in the perspective of an extension of the strain’s degradation capacity at alkaline pH. At pH 6.8 the strain grew on 2,4-D at a maximum rate (μ_max) of 0.158 h⁻¹. The half-maximum rate-associated substrate concentration (K_s) was 45 μM. At pH 8.5 μ_max was only 0.05 h⁻¹ and the substrate affinity was mucher lower than at pH 6.8. The initial attack of 2,4-D was not the limiting step at pH 8.5 as was seen from high dioxygenase activity in cells grown at this pH. High stationary 2,4-D concentrations and the fact that μ_max with dichlorprop was around 0.2 h⁻¹ at both pHs rather pointed at limited 2,4-D uptake at pH 8.5. Introduction of tfdK from D. acidovorans P4a by conjugation, coding for a 2,4-D-specific transporter resulted in improved growth on 2,4-D at pH 8.5 with μ_max of 0.147 h⁻¹ and K_s of 267 μM. Experiments with labeled substrates showed significantly enhanced 2,4-D uptake by the transconjugant TK62. This is taken as an indication of expression of the tfdK gene and proper function of the transporter. The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced the influx of 2,4-D. At a concentration of 195 μM 2,4-D, the effect amounted to 90% and 50%, respectively, with TK62 and MC1. Cloning of tfdK also improved the utilization of 2,4-D in the presence of (RS)−2,4-DP. Simultaneous and almost complete degradation of both compounds occurred in TK62 up to D = 0.23 h⁻¹ at pH 6.8 and up to D = 0.2 h⁻¹ at pH 8.5. In contrast, MC1 left 2,4-D largely unutilized even at low dilution rates when growing on herbicide mixtures at pH 8.5.     

Effect of Selected Monoterpenes on Methane Oxidation, Denitrification, and Aerobic Metabolism by Bacteria in Pure Culture

Selected monoterpenes inhibited methane oxidation by methanotrophs (Methylosinus trichosporium OB3b, Methylobacter luteus), denitrification by environmental isolates, and aerobic metabolism by several heterotrophic pure cultures. Inhibition occurred to various extents and was transient. Complete inhibition of methane oxidation by Methylosinus trichosporium OB3b with 1.1 mM (−)-α-pinene lasted for more than 2 days with a culture of optical density of 0.05 before activity resumed. Inhibition was greater under conditions under which particulate methane monooxygenase was expressed. No apparent consumption or conversion of monoterpenes by methanotrophs was detected by gas chromatography, and the reason that transient inhibition occurs is not clear. Aerobic metabolism by several heterotrophs was much less sensitive than methanotrophy was; Escherichia coli (optical density, 0.01), for example, was not affected by up to 7.3 mM (−)-α-pinene. The degree of inhibition was monoterpene and species dependent. Denitrification by isolates from a polluted sediment was not inhibited by 3.7 mM (−)-α-pinene, γ-terpinene, or β-myrcene, whereas 50 to 100% inhibition was observed for isolates from a temperate swamp soil. The inhibitory effect of monoterpenes on methane oxidation was greatest with unsaturated, cyclic hydrocarbon forms [e.g., (−)-α-pinene, (S)-(−)-limonene, (R)-(+)-limonene, and γ-terpinene]. Lower levels of inhibition occurred with oxide and alcohol derivatives [(R)-(+)-limonene oxide, α-pinene oxide, linalool, α-terpineol] and a noncyclic hydrocarbon (β-myrcene). Isomers of pinene inhibited activity to different extents. Given their natural sources, monoterpenes may be significant factors affecting bacterial activities in nature.