Microbial community structure and trichloroethylene degradation in groundwater.

Trichloroethylene (TCE) is a prevalent contaminant of groundwater that can be cometabolically degraded by indigenous microbes. Groundwater contaminated with TCE from a US Department of Energy site in Ohio was used to characterize the site-specific impact of phenol on the indigenous bacterial community for use as a possible remedial strategy. Incubations of 14C-TCE-spiked groundwater amended with phenol showed increased TCE mineralization compared with unamended groundwater. Community structure was determined using DNA directly extracted from groundwater samples. This DNA was then analyzed by amplified ribosomal DNA restriction analysis. Unique restriction fragment length polymorphisms defined operational taxonomic units that were sequenced to determine phylogeny. DNA sequence data indicated that known TCE-degrading bacteria including relatives of Variovorax and Burkholderia were present in site water. Diversity of the amplified microbial rDNA clone library was lower in phenol-amended communities than in unamended groundwater (i.e., having Shannon-Weaver diversity indices of 2.0 and 2.2, respectively). Microbial activity was higher in phenol-amended ground water as determined by measuring the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride. Thus phenol amendments to groundwater correlated with increased TCE mineralization, a decrease in diversity of the amplified microbial rDNA clone library, and increased microbial activity.

A reexamination of the substrate utilization of 2-thioorotidine-5'-monophosphate by yeast orotidine-5'-monophosphate decarboxylase.

A potential alternate substrate for orotidine-5'-monophosphate decarboxylase, 2- thio-orotidine-5'-monophosphate, was synthesized enzymatically and purified by a modification of a previous account (K. Shostak, and M. E. Jones 1992, Biochemistry 31, 12155-12161). Characterization of the product was confirmed by mass spectrometry, (31)P NMR, and utilization by orotate phosphoribosyltransferase in the direction of pyrophosphorolysis. The previous work probably did not result in the purification of the desired compound, as evidenced by our observation of 2-thioOMP's sensitivity to high temperature, as used previously. Using a very sensitive HPLC assay for the potential decarboxylated product 2-thioUMP, no measurable activity of ODCase toward the alternate substrate was observed, representing a decarboxylation rate decreased by 10(-7) from the k(cat) for ODCase toward OMP. Additionally, 2-thioOMP effects no inhibition of ODCase decarboxylation of OMP at a concentration of 50 microM, indicating a poor ability to bind to the ODCase active site. The results bear implications for proposed mechanisms for catalysis by ODCase.

Activity of yeast orotidine-5'-phosphate decarboxylase in the absence of metals.

Yeast orotidine-5'-phosphate decarboxylase was recently shown to contain zinc and to be inhibited by zinc-complexing agents. When the gene for the yeast enzyme was expressed in Escherichia coli, the gene product was devoid of metal atoms but exhibited a specific activity and molecular mass similar to those of the enzyme obtained directly from yeast. This invalidates the hypothesis that zinc is involved in substrate decarboxylation. The zinc-free enzyme undergoes thermal inactivation at a somewhat lower temperature than does the zinc-containing enzyme isolated from yeast.

Radioactivity-based and spectrophotometric assays for isoorotate decarboxylase: identification of the thymidine salvage pathway in lower eukaryotes.

A few organisms, notably some fungi, have the ability to metabolize thymidine to uracil, thus conserving the pyrimidine ring for subsequent metabolic use. Neurospora crassa possesses this pathway, termed the thymidine salvage pathway, and can utilize thymidine as a total pyrimidine source. The enzyme isoorotate decarboxylase (IDCase) completes this pathway via the enzymatic removal of the carboxylate from isoorotate to yield uracil. We describe in this communication two assays for IDCase and their application to determine activity levels, kinetic constants, and inhibitory properties. One uses [carboxy-14C]isoorotate from which the enzymatically generated 14CO2 is collected and quantitated. The second assay utilizes the spectral difference between 2-thioisoorotate and its decarboxylated product, 2-thiouracil. The spectral difference is greatest at 334 nm, out of the range of absorbance of total protein and thus usable for a spectrophotometric assay. The assays are sufficiently sensitive and accurate to be used in the measurement of Km values for both substrates. IDCase activity is found to be significantly higher in N. crassa strains lacking uc-1, a putative regulatory gene, suggesting a degree of metabolic control over this pathway. 5-Nitrouracil is found to inhibit IDCase with an estimated Ki value that is too low for accurate determination.

Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli: antibodies for orotate decarboxylation.

Antibodies capable of decarboxylating orotate were sought by immunization with a hapten designed to elicit antibodies with combining sites that resemble the orotate-binding and catalytic portion of the active site of the enzyme orotidine 5'-monophosphate (OMP) decarboxylase (orotidine-5'-monophosphate carboxy-lyase, EC 4.1.1.23). Active recombinant antibody fragments (Fabs) were selected from a combinatorial cDNA library by complementation of a pyrF strain of Escherichia coli and growth of the library-expressing cells on pyrimidine-free medium. In this biological screen, a sufficiently active antibody from the library would decarboxylate orotate to produce uracil, a pyrimidine source for the auxotroph, and would provide the cells with a growth advantage compared to cells without an active antibody. Six recombinant Fabs yielded identifiable colonies in a screen of 16,000 transformants. To enhance its stability and expression level, one of the six positive fragments was converted into single-chain form. In this form, the antibody fragment conferred a definite growth advantage to the auxotroph that was eliminated when the hapten was included in the medium. The purified single-chain antibody displayed orotate decarboxylase activity in vitro, as determined by a 14CO2 displacement assay. The specific activity of the antibody is approximately 10(-7) times that of naturally occurring OMP decarboxylase, but this antibody-catalyzed rate is estimated to be 10(8) times the background rate. The results offer the potential to use these methods to obtain catalytic antibodies for other biosynthetic reactions as well as to assess the effectiveness of the hapten transition state or active site analog in eliciting antibody catalysts.

A unique catalytic and inhibitor-binding role for Lys93 of yeast orotidylate decarboxylase.

The presence of a proton-donating catalytic amino acid side chain in orotidylate decarboxylase (ODCase) was sought by site-directed mutagenesis. Replacement of yeast ODCase Lys93 with a cysteine resulted in a mutant protein (K93C) with no measurable activity, representing a decrease in activity by a factor of, at most, 2 x 10(-8) times the activity of the wild-type enzyme. Treatment of this mutant protein with 2-bromoethylamine, designed to append Cys93 to yield S-(2-aminoethyl)cysteine, restored activity by a factor of at least 5 x 10(5) over the untreated mutant protein. Activity could not be restored by treatment with other brominated reagents designed to replace the epsilon-amino of S-(2-aminoethyl)Cys93 with a different functional group. The overall architecture of the K93C protein was not significantly changed, as judged by the similar dimerization properties (in the absence of ligands) of the mutant enzyme compared to the wild-type enzyme. The binding affinity of the substrate orotidylate was not measurably changed by the mutation, indicating that Lys93 has an essential role in catalysis which is mechanistically distinguishable from substrate binding. Apparently the mutation removes an integral portion of the active site and does not drastically affect the structural or substrate binding properties. However, the affinities of the mutant protein for the competitive inhibitors 6-azauridylate (6-azaUMP) and UMP are significantly altered from the pattern seen with the wild-type enzyme. The K93C protein has an affinity for the neutral ligand UMP which is greater than that for the anionic 6-azaUMP, in clear contrast to the preference for 6-azaUMP displayed by the wild-type enzyme. Lys93 is apparently critical for catalysis of the substrate to product and for the binding of anionic inhibitors; the data are discussed in terms of previously existing models for transition-state analogue inhibitor binding and catalysis.

Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects.

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).