Progress 04/01/04 to 03/31/09
Outputs
OUTPUTS: None. I accepted an administrative role in December, 2006 and no longer do research. PARTICIPANTS: None. I accepted an administrative role in December, 2006 and no longer do research. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
None. I accepted an administrative role in December, 2006 and no longer do research.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: The undergraduates working in my laboratory did manage during a second round of directed mutagenesis to select two clones of the original NADP-utilizing mutant that coded for formate dehydrogenase activity with a lower Km for NADP than isolated during the first round. However, in December I gave up my research appointment to become an Associate Dean in the College of Agricultural Sciences and Natural Resources and the research was discontinued.
PARTICIPANTS: Brittany Prather worked on this project as an undergraduate and is now in the Molecular Biology Ph.D. program at the University of Iowa. Josh Widhalm worked on this project as an undergraduate and is now in the Agronomy Ph.D. program at the University of Nebraska.
Impacts
This research demonstrated that it is feasible to develop a formate dehydrogenase enzyme that will utilize NADP as a cofactor. The hypothesis that such an enzyme placed int he plant mitochondrion would fix carbon dioxide into formate has not yet been tested.
Publications
- M. Zeece, J. Markwell, G. Sarath and X. Gu (2006) Proteomic assessment of allergens in food. In S. Koppelman and S.L. Hefle, eds, Detecting Allergens in Food, Woodhead Publishing Ltd, Cambridge.
- B.J.S.C. Olson and J. Markwell (2007) Assay of protein concentration. Curr. Protocol. Prot. Sci., 3.4.1-3.4.29.
- B.L. Prather, J.R. Widhalm, J. Markwell and P.L. Herman (2006) Development of a System for Directed Evolution of Arabidopsis Formate Dehydrogenase to Utilize NADP as a Cofactor. Rev. Undergrad. Res. Agric. Life Sci. 1: on-line publication available at http://digitalcommons.unl.edu/rurals/vol1/iss1/3
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Progress 10/01/05 to 09/30/06
Outputs
The major research effort involved study of leaf formate dehydrogenase (FDH; EC 1.2.1.2), an NAD+-dependent enzyme that catalyzes the oxidation of formate to CO2 in the mitochondria of higher plants. The enzyme is specific for its NAD+ coenzyme and does not utilize NADP+ for the oxidation of formate. Interestingly, once the FDH has been heated at 60C for five minutes and cooled, it is able to use NADP+ as a substrate for the oxidation of formate. This ability of FDH to assume a conformation able to use NADP+ in a mitochondrial compartment is of interest. The plant mitochondrion has ratios of NADPH/NADP+ greater than unity and the presence of an NADP+-specific FDH could potentially provide a second source for CO2 fixation. We are attempting to produce a FDH enzyme with a high affinity for NADP as a cofactor using directed evolution, We had previously cloned the cDNA for the Arabidopsis FDH protein without the transit sequence into a pETBlue vector for directed
mutagenesis and managed to get this construct to express active recombinant FDH in E. coli Tuner cells in a constitutive fashion. This clone was designated pFDH-0. The conditions for random mutagenesis by inclusion of Mn2+ in error-prone PCR were established, and we are able to produce an average of one to two base changes per kb of PCR product. During the past year, our efforts have been largely spent on developing the methodology for screening that would permit us to identify the few mutants in the first mutagenesis round that had acquired the ability to utilize NADP as a cofactor. This was accomplished by two undergraduates in my laboratory. To date, we have screened over a thousand putative mutants from the initial random mutagenesis and have isolated three that have clear NADP+ activity. For the pFDH-18 mutant, the Km for formate remains about 10 mM and that for NAD+ has been raised from 65 micro-M to 0.18 mM, with the new Km for NADP+ being 2.5 mM. The other two mutants have
kinetics that were unexpected. Rather than the linear Lineweaver-Burk plots observed for the wild-type, pFDH-0 and pFDH-18 strains, these mutants have repeatedly produced Lineweaver-Burk plots that are concave-downward. Our interpretation of this phenomenon is that the two subunits of the homodimeric enzyme are not folding identically. When cultures of E. coli containing the plasmids for these two mutants were incubated at 15C for two days, the resulting enzymes produced Lineweaver-Burk plots that were linear. These results are consistent with our previous observations that the kinetic characteristics of the enzyme isolated from the plant vary with the treatment during isolation, but raise the question of whether the in vivo properties of the enzyme may also vary with environmental conditions. During the next year, we intend to conduct a second round of random mutagenesis on the three NADP-utilizing mutants we have isolated and screen for the ability to utilize this cofactor with an
increased affinity
Impacts
These results constitute a proof-of-concept for our ability to use directed evolution to produce a variant FDH able to utilize NADP as a cofactor. This is the first step in the process of finding out whether an NADP-FDH could be put into the plant mitochondrion and have a positive effect on photosynthetic efficiency.
Publications
- B.L. Prather, J.R. Widhalm, J. Markwell and P.L. Herman. 2006. Development of a System for Directed Evolution of Arabidopsis Formate Dehydrogenase to Utilize NADP as a Cofactor. Rev. Undergrad. Res. Agric. Life Sci., in press.
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Progress 10/01/04 to 09/30/05
Outputs
During the past year, we have initiated the directed evolution of a FDH enzyme able to utilize NADP as a cofactor. We have cloned the cDNA for the Arabidopsis thaliana FDH protein without the transit sequence into a pETBlue vector for directed mutagenesis and managed to get this construct to express active FDH in E. coli Tuner cells in a constitutive fashion. This clone has been designated pFDH-0. The conditions for random mutagenesis by inclusion of Mn2+ in error-prone PCR have been established and we are able to produce an average of one to two base changes per PCR product. The presence of mutations has been validated with the Transgenomic Surveyor nuclease system. We are able to transform the FDH construct in the pETBlue vector into E. coli and get expression of FDH activity in colonies grown in microtiter plates. When we have conducted blue/white screening, we routinely produce over 90% white colonies, so we are able to clone the mutated plasmids directly into the
expression vectors. The challenge so far has been in the screening of putative mutant lines for altered FDH activity in microtiter plates. Because of the high scatterance of the lysates at UV wavelengths (e.g. 340 nm), we attempted an assay coupling NAD+ and NADP+ reduction to DCIP and PMS. After much effort, we gave up on this system because of the high intial absorbance and its negative effect on signal to noise ratio. We have since combined cell lysis with a proprietary surfactant and extensive centrifugation in microplates to direct spectrophotometric measurement of NAD+ and NADP+ reduction at 340 nm. This has proved more satisfactory and we were able to screen two 96 well plates with pFDH-0 and observe 27.8 mU/mg +/- 0.58 (sem) NAD+-FDH activity in 174 wells. Thus, we have all the pieces in place to begin production of a library of random mutants of the Arabidopsis FDH and screen several thousand members of the library for the ability to utilize NADP as a cofactor in the
oxidation of formate. To date, we have screened over 700 putative mutants and have isolated one that has clear NADP+ activity. The Km for formate remains about 10 mM and that for NAD+ has been raised from 65 uM to 2.3 mM and the new Km for NADP+ is 7 mM. We intend to continue to screen approximately 10,000 mutants of the pFDH-0 clone, and isolate further mutants. Once we have isolated such mutants, further rounds of random mutagenesis will be used for directed evolution of variants with higher affinities for the substrates in both the forward and reverse directions.
Impacts
If we are able to produce an FDH with a high affinity for NADPH by directed evolution, we will use it to transform plants and express the enzyme in mitochondria. This will permit us to test whether the presence of such an enzyme may cause a second site for carbon dioxide formation in plant leaves and alter photosyntheic efficiency.
Publications
- P. Xiang, E.J. Haas, M.G. Zeece, J. Markwell and G. Sarath (2004) C-terminal 23 kDa polypeptide of soybean Gly m Bd 28 K is a potential allergen. Planta, 220: 56-63.
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Progress 04/01/04 to 09/30/04
Outputs
During the past year, we have initiated the directed evolution of a FDH enzyme able to utilize NADP as a cofactor. We have succeeded in developing conditions for expression of active FDH in Escherichia coli to a specific activity of over 1 U/mg protein using the pET28 vector. We have cloned the cDNA for the Arabidopsis thaliana FDH protein without the transit sequence into a pETBlue vector for directed mutagenesis. The conditions for random mutagenesis by inclusion of Mn2+ in error-prone PCR have been established and we are able to produce an average of one to two base changes per PCR product. The presence of mutations has been validated with the Transgenomic Surveyor nuclease system. We are able to transform the FDH construct in the pETBlue vector into E. coli and get expression of FDH activity in colonies grown in microtiter plates. Thus, we have all the pieces in place to begin production of a library of random mutants of the Arabidopsis FDH and screen several
thousand members of the library for the ability to utilize NADP as a cofactor in the oxidation of formate. Once we have isolated such mutants, further rounds of random mutagenesis will be used for directed evolution of variants with higher affinities for the substrates in both the forward and reverse directions.
Impacts
Leaf formate dehydrogenase (FDH) is an NAD-dependent enzyme that catalyzes the oxidation of formate to CO2 in the mitochondria of higher plants. Sequence analysis and other preliminary experiments suggested that FDH might also be targeted to the chloroplasts of Arabidopsis thaliana and other plant species. Kinetic analyses carried out to confirm the kinetics and substrate specificity of the Arabidopsis FDH revealed several anomalies. Although the enzyme is specific for its NAD cofactor and did not utilize NADP+ for the oxidation of formate, NADPH appeared to be an inhibitor of the NAD+-dependent formate oxidation. Once FDH has been heated at 60C for five minutes and cooled, it is able to use NADP+ as a substrate for the oxidation of formate. This ability to assume a conformation able to use NADP+ in a chloroplastic compartment is of interest. The chloroplastic presence of such an enzyme could potentially provide a second source for CO2 fixation. This project will
attempt to place a NADP-utilizing FDH into the chloroplast and determine if it will fix CO2.
Publications
- R. Roychaudhuri, G. Sarath, M. Zeece and J. Markwell (2004) Stability of the allergenic soybean Kunitz trypsin inhibitor. Biochim. Biophys. Acta, 1699: 207-212.
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