Progress 10/01/05 to 09/30/08
Outputs
OUTPUTS: Cinnamyl alcohol dehydrogenases of Burkholderia cepacia and Escherichia coli were overexpressed in E. coli and purified to homogeneity using ammonium sulfate fractionation, gel exclusion chromatography and dye ligand chromatography. They were assayed by NADP+ reduction by cinnamyl alcohol at pH 9.0, following absorbance at 340 nm. Substrate specificity was studied for alcohols and a few aldehydes. A knock-out strain of B. cepacia, in which the gene was disrupted by insertion of a kanamycin acetyltransferase gene, was constructed, and the effect of the knock-out assessed on minimal phthalate medium and at inhibitory cinnamaldehyde levels. Nitroaromatic reductases cloned from Ralstonia pickettii YH105, R. eutropha JMP134 and Pseudomonas pseudoalcaligenes JS45 (hereafter referred to by the strain numbers) and expressed in E. coli were purified, using ammonium sulfate fractionation and gel exclusion, hydroxylapatite and ion exchange chromatography. The JMP134 and JS45 enzymes were purified to homogeneity, the YH105 enzyme is not yet pure. They were assayed by oxidation of NADPH, observed by decrease of absorbance at 340 nm. Substrate specificity (Km and Vmax) was surveyed. The kinetic mechanisms were studied by varying concentrations of substrates and product and dead-end inhibitors. Inorganic anions (chromate, molybdate, selenate) were studied as substrates. A spectrophotometric assay for Cr+++, extracting the 8-hydroxyquinoline complex into methyl isobutyl ketone and measuring absorbance at 417 nm, was devised, in order to measure stoichiometry of chromate reduction. Hydroxylaminobenzoate lyase of YH105 was expressed in E. coli and partially purified. p-Hydroxylaminobenzoate and analogs were synthesized as substrates, and an assay developed using high performance liquid chromatography to separate product protocatechuate from substrate. The Km for p-hydroxylaminobenzoate was determined, and substrate analogs surveyed as substrates. Results have so far been reported only in a poster at the 1st annual Microbiology Symposium, Dept. of Biochemistry and Microbiology, SEBS, Rutgers; papers are in preparation. PARTICIPANTS: Dr. Theodore Chase, Jr. officially retired 7/1/07. Graduate student Farley Hunter completed the M.S. degree in January 2008. TARGET AUDIENCES: Papers will be prepared for publication in scientific journals. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Cinnamyl alcohol dehydrogenases of B. cepacia and E. coli have sequential kinetic mechanisms. Cinnamyl alcohol and trans-2-hexenol are the best alcohol substrates (pH optimum 9.0), but benzaldehyde is a better substrate (higher Vmax/Km) than cinnamaldehyde for the B. cepacia enzyme and equivalent for the E. coli enzyme (pH optimum 6.8). The knock-out strain of B. cepacia, with the gene for the enzyme disrupted, grew as well as wild-type on phthalate minimal medium, and was no more sensitive to inhibition of growth by cinnamaldehyde. The biological function of the enzyme is thus unknown. The JS45 and JMP134 nitroaromatic reductases have been purified to homogeneity. The latter is more stable at pH 8.0 rather than 6.8. Both are flavoenzymes, as shown by yellow color and spectral analysis. The YH105 enzyme has not yet been purified to homogeneity. Substrate survey of the JMP134 and YH105 enzymes showed their best substrates (highest Vmax/Km) to be those on which the organisms were isolated, 3-nitrophenol and p-nitrobenzoate respectively. Nitrobenzene and substrates with nonpolar p-substituents have Kms for the JS45 enzyme too low to be determined (< 5 micromolar), while the Km for NADPH is too high to be determined accurately (> 1 mM). Kinetic studies with o-nitrotoluene as substrate indicated a ping-pong mechanism, in which NADPH binds, reduces the bound FMN, and product NADP+ dissociates before the nitro substrate binds. p-Hydroxylaminobenzoate and -benzenesulfonamide are uncompetitive inhibitors vs. the corresponding nitro substrates, as expected if they dissociate before NADP+. Vmax values for most substrates are similar (ca. 5 micromole/min per mg), suggesting that flavin reduction is the rate limiting step. Benzaldehyde and p-nitrobenzaldehyde (a very poor substrate) are inhibitors, non-competitive vs nitrobenzene rather than competitive. Kinetic studies of the YH105 and JMP134 enzymes indicate a sequential mechanism, in which the nitro substrate binds before release of NADP+ product, and the second NADP+ product is last off (competitive product inhibitor vs NADPH). p-Nitrobenzoate shows substrate inhibition of YH105, competitive vs. NADPH, at concentrations above 0.25 mM. All 3 enzymes reduce chromate efficiently, but not molybdate or selenate. Limiting Kms for chromate are .065 mM for JMP134, .0245 mM for JS45. Vmax for JMP134 with chromate is 1.9x that with 3-nitrophenol. Vmax for JS45 cannot be compared because of the high Km for NADPH, but at 0.1 mM NADPH activity on chromate is 45% of that on nitrobenzene. Two NADPH are oxidized per chromate reduced. These organisms therefore potentially could be used to reduce chromate to Cr+++ in the environment, reducing toxicity and solubility. p-Hydroxylaminobenzoate lyase of YH105 requires strong reducing conditions for stability and activity. The Km for p-hydroxylaminobenzoate is 0.079 mM. 3-Methyl-4-nitrobenzoate is also a substrate, but not 3-nitrobenzoate or 4-nitrophenylacetate. Thus though specificity of the nitroaromatic reductase is broad, greater specificity of the lyase limits usefulness of the organism for degradation of other nitroaromatics in the environment.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: p-Hydroxylaminobenzoate lyase of Ralstonia (formerly Pseudomonas) pickettii, cloned in Escherichia coli, was partially purified, using ammonium sulfate precipitation, gel filtration and ion exchange chromatography. It was assayed using high performance liquid chromatography to separate substrate and product. The enzyme requires strong reducing conditions for stability and activity; its instability limited the purification achieved. The Michaelis constant for p-hydroxylaminobenzoate is 0.079 mM. m-Hydroxylaminobenzoic acid, p-hydroxylaminophenylacetic acid and 3-methyl-4-hydroxylaminobenzoic acid were synthesized, by the same procedure used for p-hydroxylaminobenzoic acid. However, only 3-methyl-4-hydroxylaminobenzoate was a substrate for the enzyme, and a poor one. A spectrophotometric method for determination of Cr+3 was devised, for measurement of this product of reduction of chromate by nitroaromatic reductases. It involves complexation with 8-hydroxyquinoline and
extraction of the complex into methyl isobutyl ketone, in which the molar extinction coefficient of the complex is 4,800 L/mole cm at 417 nm. It is necessary to heat the reaction at 95 C for 1 hr to complete replacement of coordinated water by 8-hydroxyquinoline. The three nitroaromatic reductases under study all reduce chromate effectively with a stoichiometry of 2 NADPH oxidized:1 chromate reduced. Benzaldehyde and p-nitrobenzaldehyde (a very poor substrate) were inhibitors of JS45 nitroaromatic reductase, but non-competitive vs. nitrobenzene rather than competitive; benzaldehyde was also non-competitive vs. NADPH. A new purification of reductase JMP134, cloned from Ralstonia eutropha into E. coli, using a higher pH buffer, yielded nearly pure enzyme with much greater retention of bound flavin mononucleotide.
PARTICIPANTS: Theodore Chase, Jr., Ph.D., PI Farley A. Hunter (completed M.S. on this project) Irene Wohlman, B.S.
Impacts
p-Hydroxylaminobenzoate lyase is much more substrate-specific than the reductase preceding it in the pathway of biodegradation of p-nitrobenzoate, limiting the use of the organism in biodegradation of other nitroaromatic compounds in the environment. Bacteria expressing nitroaromatic reductases may be effective in reducing toxic chromium +6 (chromate) in the environment, by reducing it to the less toxic and less soluble chromium +3 ion.
Publications
- Purification and characterization of the hydroxylaminobenzoate lyase from Pseudomonas pickettii YH105, cloned in Escherichia coli. Farley A. Hunter. M.S. thesis, The Graduate Schools, Rutgers - The State University of New Jersey and University of Medicine and Dentistry of New Jersey, December 2007.
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Progress 01/01/06 to 12/31/06
Outputs
The Kms of Pseudomonas pseudoalcaligenes JS45 nitrobenzene reductase for nitrobenzene and other nonpolar substrates are very low, < 5 micromolar. Km and Vmax values have been determined for o-nitrotoluene, o-, m- and p-nitrobenzoate (NBz), p-nitrophenylacetate, p-nitrobenzenesulfonamide, p-nitrobenzenesulfonic acid, and p-nitroaniline. Vmax values for the nonpolar substrates and the better polar substrates are around 12.5 micromoles/min per mg protein at [NADPH] = 0.3 mM, lower for o-NBz and p-nitroaniline. p-NBz and p-nitrophenylacetate show substrate inhibition. Lineweaver-Burk plots of 1/v vs 1/[o-nitrotoluene] at changing [NADPH] are parallel, indicating a ping-pong mechanism: NADPH binds and reduces enzyme-bound FMN, then departs as NADP+, followed by binding of substrate and its reduction to the nitroso compound. NADPH binds again, reduces FMN again, which reduces the nitroso intermediate to the final hydroxylamino product. Inhibition by hydroxylamino
(NHOH-)benzoate and NHOH-benzenesulfonamide is uncompetitive vs. the nitro substrates, as expected if the reductions are irreversible. Product inhibition by NADP+ is competitive vs. nitroaromatic substrate, as expected if it dissociates from the FMNH2 enzyme form which then binds substrate, and competitive vs NADPH, as expected if it is the last product off (and binds to free enzyme). Replot of Vmax at varying [o-nitrotoluene] vs. NADPH concentration (0.2-1.0 mM) indicates a Km for NADPH > 1 mM. However, since in ping-pong mechanisms the Km for one substrate decreases with decreasing concentration of the other, the Km for NADPH at low environmental concentrations of nitroaromatic compounds is likely to be lower. The sequential kinetic mechanism of p-NBz reductase of Ralstonia pickettii YH105 has also been studied. NADP+ as product inhibitor is competitive vs. NADPH, non-competitive vs p-NBz. NHOH-benzoate is uncompetitive vs NBz, noncompetitive vs NADPH. The substrate analog
terephthalate is competitive vs NADPH, like substrate inhibition by p-NBz, while the non-substrate p-nitroaniline is uncompetitive vs NBz, may bind to E-NADP+. These results are consistent with a mechanism where NADPH binds first, then p-NBz, followed by reduction of the latter to nitrosobenzoate and dissociation of NADP+. A second NADPH binds and a second reduction occurs. NHOH-benzoate dissociates, then NADP+ (as shown by the non-competitive inhibition vs. NADPH). Substrate inhibition by p-NBz would be due to unproductive binding to free enzyme. Chromate is a substrate for all three nitroaromatic reductases. The purification of hydroxylaminobenzoate lyase of YH105 has been considerably improved, with introduction of a hydroxylapatite adsorption step. Substrate (alcohols, aldehydes) specificity of the cinnamyl alcohol dehydrogenase of Escherichia coli has been studied.
Impacts
The several enzymes studied differ not only in what polluting compounds they act on, but in how they do it. This will suggest which organisms might best be used to clean up a specific polluting compound. They also can detoxify other polluting compounds, as by reducing chromate to the less toxic, less soluble Cr(III) ion, reducing its hazard.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
It is too early in the life of the project to report at this time.
Impacts
A study of how microorganisms break down organic chemical compounds which can pollute the environment. The comparison of enzymes from several microrganisms which reduce nitroaromatic compounds shows how they differ in various respects, and could be combined to remove mixtures of such compounds from the environment. They may also detoxify other pollutants such as chromate.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The nitroaromatic reductases ofRalstonia pickettii YH105 and Pseudomonas pseudoalcaligenes JS45, expressed in E. coli, have been purified to near homogeneity. Kinetic study of the YH105 reductase, varying concentrations of both 4-nitrobenzoate and NADPH, showed a sequential mechanism (intersecting plots of 1/v vs. 1/NADPH]) and substrate inhibition at [4-nitrobenzoate] above 0.25 mM. This contrasts with known nitroaromatic reductases such as that of Escherichia coli, which have ping-pong mechanisms, NADPH reducing the bound FMN which subsequently reduces the substrate. The limiting Michaelis constant for 4-nitrobenzoate is 0.0326+/-0.004 mM, and for NADPH 0.0154+/-.00375 mM. The substrate inhibition is competitive vs. NADPH, i.e. slopes of plots of 1/v vs. 1/[NADPH] increase again at higher 4-nitrobenzoate concentrations. The nitroaromatic reductase of Ralstonia eutropha JMP134 similarly shows a sequential mechanism, limiting Michaelis constant for 3-nitrophenol =
2.43+/-0.14 micromolar, for NADPH 9.61+/-1.3 micromolar. Comparison of 4-nitrophenol and 4-nitrosophenol (the intermediate product/substrate of the 4-electron reduction) at pH 5.8 (where both are predominantly neutral) also showed a sequential mechanism, 4-nitrosphenol having a much lower Michaelis constant, and both showing substrate inhibition above 0.25 mM. Thus substrate inhibition is not a feature only of the four-electron, two-NADPH reduction. The purified YH105 enzyme appears to be active without FMN, unlike other nitroaromatic reductases (including those of JS45 and JMP134). Kinetic study of the JS45 enzyme has not proceeded as far (the Michaelis constant for NADPH is high, above 0.2 mM, and for nitrobenzene very low), but it has been found that the enzyme is five times as active in phosphate buffer as in MOPS (3-morpholinopropanesulfonic acid). The other enzymes show slightly higher activity in phosphate. The hydroxylaminobenzoate lyase of YH105, like that of Comamonas
acidovorans NBA-10 (Groenewegen, P.E.J., and de Bont, J.A.M., Arch. Microbiol. 158:381-6 [1992]) is stabilized by NADH. In an attempt to find the function of cinnamyl alcohol dehydrogenase of Burkholderia cepacia DBO-1 (a similar gene is found in E. coli and other microrganisms), we have been trying to knock out the gene by insertion of a kanamycin resistance cassette. At least one mutant has been obtained. It grows on aromatic substrates (phthalate, coumarate, benzyl alcohol, phenylalanine) only when supplemented with yeast extract, unlike the wild type organism, suggesting a possible role in coenzyme biosynthesis.
Impacts
The comparison of enzymes from several microrganisms which reduce nitroaromatic compounds shows how they differ in various respects, and could be combined to remove mixtures of such compounds from the environment.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The nitroaromatic reductase of Ralstonia eutropha JMP134, expressed in E. coli, has been purified by ammonium sulfate fractionation, gel filtration (which indicated it to be a monomer of mol. wt. 30,000 Da) and ion exchange chromatography. It contains a bound flavin coenzyme, presumably FMN. NADPH is the electron donor; NADH is not used. Among a wide variety of nitroaromatic substrates, 3-nitrophenol is the best (kcat/Km = 8.32/min x micromolar) while 4-nitrophenol is the worst (kcat/Km = 0.04/min x micromolar) due to a high Km. Nitroso compounds, intermediates in the reduction, showed high Vmax and lower Kms than the corresponding nitro compounds, as expected if they are tightly bound intermediates. 2-Nitro- and nitroso-1-naphthol showed substrate inhibition, while methyl 4-nitrobenzoate showed substrate activation. Vmax is determined largely by the redox potential of the substrate, being favored by electron withdrawing substituents, while specificity is determined
by Km; but 3-nitrophenol, the growth substrate on which the organism was isolated, shows the highest Vmax as well as a low Km, The nitroaromatic reductase of Ralstonia pickettii YH105 has also been purified, though in poor yield due to lability at high ionic strength, believed to be due to loss of the FMN cofactor. The nitroaromatic reductases have been expressed with a six-histidine C-terminal tag, but do not adsorb to immobilized Ni columns.
Impacts
This information will help define how different microorganisms can be used to remediate different pollutants in the environment (in this case, 3-nitrophenol and other nitroaromatic compounds).
Publications
- Purification and Characterization of the 3-Nitrophenol Reductase from Ralstonia eutropha JMP134, Cloned in Escherichia coli. J.S. Campor. M.S. Thesis, Rutgers University, October 2003.
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Progress 01/01/02 to 12/31/02
Outputs
Vmax and Km values were determined for 25 alcohol substrates of Burkholderia cepacia cinnamyl alcohol dehydrogenase, cloned in E. coli and purified. Cinnamyl alcohol and trans-2-hexenol were the best substrates (highest Vmax/Km ratio), as expected from the equilibrium-shifting effect of the 2,3 double bond which conjugates with the product carbonyl group. Substrate inhibition was seen at higher concentrations of most substrates. The genes for nitroaromatic reductases of Ralstonia pickettii YH105 and Pseudomonas pseudoalcaligenes JS45 were transferred to His-tagged vectors and expressed in E. coli; the reductase of Ralstonia eutropha JMP134 was also expressed, without His-tag, and purification was begun. An hplc assay for hydroxylaminobenzoate lyase was devised and tested, and activity of the B. cepacia enzyme, expressed in E. coli, was found to be optimal at 25 degree, pH 7.3.
Impacts
An assay for hydroxylaminobenzoate lyase was devised and tested, and activity of the B. cepacia enzyme, expressed in E. coli, was found to be optimal at 25 degree, pH 7.3. This information aids our understanding of how E. coli functions.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The gene for the constitutively expressed cinnamyl alcohol dehydrogenase of Escherichia coli, yahK in the genome sequence, was cloned by PCR amplification, topoisomerase-catalyzed incorporation into vector pTrcHis, and transformation of E. coli. This protein and the cinnamyl alcohol dehydrogenase of Burkholderia cepacia, also cloned in E. coli, have been purified essentially to homogeneity by ammonium sulfate precipitation, gel exclusion and AffiGel Red chromatography. However, the B. cepacia enzyme loses much activity during gel filtration, possibly due to subunit dissociation. The p-nitrobenzoate reductase of Ralstonia pickettii YH 105 has been expressed in E. coli; however, substrate specificity studies were complicated by presence of native E. coli nitroaromatic reductase. The Ralstonia enzyme has highest activity on p-nitrobenzenesulfonate, p-nitrobenzene-sulfonamide, p-nitrobenzoate and 5-nitrofuroate, and no activity on menadione, a substrate of the E. coli
enzyme. The plasmid bearing the R. pickettii gene was transferred to E. coli NFR343, which lacks the indigenous reductase. Enzyme purification studies have been slowed by sensitivity of the enzyme to ammonium sulfate. An assay for catechols was studied as an assay for the activity of p-hydroxylaminobenzoate lyase, also cloned from R. pickettii, which forms 3,4-dihydroxybenzoate. However, because Fe+++, chelated and reduced by catechols, is also reduced by SH compounds needed for the enzymatic reaction, and to some extent by hydroxylaminobenzoate, this procedure was abandoned. Currently an hplc assay is under study.
Impacts
(N/A)
Publications
- J. Campor, G.J. Zylstra and T. Chase. 2001. Substrate Specificity of Cloned p-Nitrobenzoate Reductase of Ralstonia pickettii YH105. Annual Mtg. N.J. Academy of Sciences
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