Topic: Research Proposal Essay Example

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Topic: Research Proposal

Aims and background

The enzyme Indole-3-pyruvate dehydrogenase/hydropyruvate dehydrogenase/3-(4-hydroxyphenyl) pyruvate dehydrogenase has been shown to catalyse the first step in an alternative route for indolepyruvate to auxin. The specificity of this enzyme has not been investigated although it is thought to catalyze the reduction of indolepyruvate in indole-3-lactase. Another enzyme that catalyses the same reaction but with a different substrate has been identified in a number of plant tissues including endosperm. This experiment aims to determine whether NADPH/NADH-dependent reduction of indole pyruvate can be detected and compare reaction rate with reduction of hydroxypyruvate and 3-(4-hydroxyphenyl) pyruvate.

Curtis and Leszek (1992) revealed that hydroxypyruvate and glyoxylate enzymes are the main metabolites involved in metabolite photorespiration. The distribution of these enzymes varies in leaves and peroxisomes. Cytosolic glyoxylate reductase is the main reducing enzyme in leaves. On the other hand, hydroxypyruvate reductase is highly active in peroxisomes and uses NADH as the preferred cofactor (Reumann, 2000). However, NADPH can also be used by the peroxisomal enzyme though to a lesser extend. In the cytosol, a second hydroxypyruvate reductase exists. Cytosolic hydropyruvate enzyme preferentially uses NADPH and to a lesser extends NADH as cofactors.

However, the assay cannot distinguish between dehydrogenase activity and reaction hypothesized for YUCCA FMO. Similarly, indole-3-pyruvate is highly unstable hence the need to check the stability of the enzyme with all assay components prior to carrying out the assay. The pre-incubation buffer will also be included in determination of the enzyme stability.

Research project

Research literature shows that photorespiration is a critical pathway in plant metabolism. The process occurs when plant stomata are closed to prevent water loss during hot and dry days (Tolbert et al 2004). As a result, photorespiration can result to net loss of nitrogen and carbon and lead to slowed growth. Arguably, the removal of carbon molecules from the Calvin cycle lowers the efficiency of photosynthesis although the specific functions remain controversial. When the carbon dioxide levels drop below 50 ppm within the leaves, Rubisco starts to combine oxygen molecules with RUBP instead of the usual carbon dioxide. This results to production of phosphoglycolate a toxic 2-carbon molecule instead of 2 3-carbon PGA. In response, the plant must remove the phophoglycolate by converting the phosphate group to glycolic acid and transporting the product to the peroxisome for further conversion to glycine. Glycine is converted into serine in the mitochondria and subsequently used to make other organic compounds.

The photorepiratory cycle is regulated by enzymes. During decarboxylation, pyruvate dehydrogenase complex (PDC) catalyses the conversion of pyruvate into acetyl-coA. The Acetyl-coA is important in cellular respiration in the citric acid cycle (Zelitch, 2007). However, the deletion of any of the core enzymes of the photorespiratory cycle, with the exception of hydroxypyruvate reductase (HPR1) results to severe air-sensitivity of the mutants.

Kleczkoowski et al (1991) revealed that hydroxypyruvate reductase provides a cystosolic bypass to the photorespiratory corecycle in Arabidopsis thaliana. The deletion of this enzyme results in elevetd levels of hydroxypyruvate among other metabolites in leaves. However, the combined deletion of HPR1 and HPR2 results in distinct air-sensitivity and significant decrease in photosynthetic performance.

Timm et al (2008) suggests that the photorespiration mechanism is not confined to mitochondria, peroxisomes and chloroplasts but also to the cytosol. Nevertheless, the level to which Cytosolic reactions contribute to the photo respiratory process in different natural environments is not well documented. However, Timm et al suggest that it could be regulated by peroxisomal redox homeostasis and dependent on the availability of NADH.

The reduction of hydropyruvate in leaves is achieved by the catalytic activity of NADH and NADPH. NADH enzyme prefers hydroxypyruvate reductase (HPR-1). NADPH prefers HPR-2 or glyoxylate reductase (GR-1). In a research by Leszek et al they showed that NADPH-preferring hydroxypyruvate reductase (HPR-2) was un-competitively inhibited by oxalate, an end product of metabolism in plants. The glyoxylate and hydroxypyruvate-dependent rates of the HPR-2 enzymes were affected. Oxalate could therefore significantly regulate extra-peroxisomal metabolism of hydropyruvate and glyoxylate in photosynthetic tissues (Scheibe et al, 2005).

Spaepen et al (2007) described the ability of bacterial species to produce indole-3-acetic acid (IAA) an auxin phytohormone. In the study, the authors discussed recent data and merging views on IAA as a microbial metabolic and signaling molecule. The researchers argue that different biosynthesis pathways have redundancy among plant-associated bacteria. IAA-producing bacteria and plant interact resulting to varying pathogenesis and phytostimulation. The scholars highlighted the role of bacterial IAA in different microorganism-plant interactions. In bacteria, IAA can also serve as a signaling molecule hence has a direct effect on bacterial physiology.

It is assumed that the conversion of hydroxypyruvate to glycerate by HPR1 relies on exclusive peroxisome-localized photo respiratory complex (Boldt et al, 2005). The hpr1 mutants show relative air-sensitivity. The researchers used genetically defined knockout mutants to show that photo respiratory carbon flux can efficiently one of the critical reactions in the cycle.

Indole-3-pyruvate dehydrogenase could catalyze the firts step in an alternative pathway for indolepyruvate to auxin (Schwarte and Bauwe, 2007). Hydroxyphenylpyruvate dehydrogenase which catalyze the same reaction but using a different substrate have been identified in a number of plant tissues including endosperm. The substrate specificity of hydroxypyruvate has not been investigated. This experiment will determine whether NADPH/NADH-dependent reduction of indole pyruvate can be detected. Further, the lab will compare the reaction rate with reduction of hydroxypyruvate and 3-(4-hydroxyphenyl) pyruvate.

Study design

The experiment will determine whether NADPH/NADH-dependent reduction of indole pyruvate can be detected. Further, the lab will compare the reaction rate with reduction of hydroxypyruvate and 3-(4-hydroxyphenyl) pyruvate.

To achieve this, an extraction buffer will be used consisting of 40 mM tricin pH 7.8, 2mM Mgvl2, 1mMedta, 14mM mercaprotoethanol, Ammonium sulfate of 45-60% saturation, Cofactors NADPH and NADH.

The reaction catalysed is hydroxypyruvate + NADPH+ H+ > D-glycerate + NADP+ 3-(4-hydroxyphenyl) pyruvate + NADPH + H+ > ®-3-(4-hydroxyphenyl) lactate + NADP+ INDOLE-3-PYRUVATE + NADPH + NADPH +H+ > INDOLE-3-LACTATE +NADP+

We will determine the catalytic activity with Beta-hydroxypyruvate, Hydroxyphenylpyruvate and glyoxylate which have been measured with different extraction preparations but probably the same enzyme. There is no available information on optimum temperature on the catalytic reaction or the need for reducing agents. However, it is predicted that the molecular weight of 70 kDa will be 34.26 X 2. The assay conditions will be maintained at a pH of 6.5 by using 10Mm and 0.2mM NADPH and substrate. We predict that the PI will measure 5.85. The hypothesis is that NADPH/NADH-dependent reduction of indole pyruvate will be detected by comparing rate of reaction with reduction of hydroxypyruvate and 3-(4-hydroxyphenyl) pyruvate. To test this hypothesis, data will be analyzed using Chi Square at 0.05 confidence level to compare expected and observed measurements.

References

Boldt, R., Edner, C., Kolukisaoglu, Ü. Hagemann, M., Weckwerth, W., Wienkoop, S., Morgenthal, K., and Bauwe, H. (2005). D-Glycerate 3-kinase, the last unknown enzyme in the photorespiratory cycle in Arabidopsis, belongs to a novel kinase family. Plant Cell, 17 2413–2420.

Curtis, G. & Leszek, K. (1992). The enzymatic reduction of glyoxylate and hydroxypyruvate in leaves of higher plants. Plant Physiology, 100(2): 552-556.

Kleczkoowski, L. Randall, D. & Edwards, G. (1991). oxalate as a potent and selective inhibitor of spinach (spinacia oleracea) leaf NADPH-dependent hydroxypyruvate reductase. Biochemistry Journal, 276, 125-127.

Reumann, S. (2000). The structural properties of plant peroxisomes and their metabolic significance. Biological. Chemistry. 381 639–648.

Scheibe, R., Backhausen, J.E., Emmerlich, V., and Holtgrefe, S. (2005). Strategies to maintain redox homeostasis during photosynthesis under changing conditions. Journal of Experimental Botany. 56 1481–1489.

Schwarte, S. & Bauwe, H. (2007). Identification of the photorespiratory 2-phosphoglycolate phosphatase, PGLP1, in Arabidopsis. Plant Physiology. 144 1580–1586.

Spaepen, S. Vanderleyden, J. & Remans, R. (2007). indole-3-acetic acid in microbial and microorganism-plant signaling. Federation of European Microbiological Societies, 31:425-448.

Timm, S. Nunes-Ness, A. Parnik, T. Morgenthal, K. Wienkoop, S. Keerberg, O. Weckwerth, W. Kleczkowski, L. Fernie, A. & Bauwe, H. (2008). A cytosolic pathway for the conversion of Hydroxypyruvate to Glycerate during photorespiration in Arabidopsis. The plant Cell, Vol. 20(10): 2848-2859.

Tolbert, E. Yamazaki, K. & Oeser, A. (2004). Localization and properties of hydroxypyruvate and glyoxylate reductases in spinach leaf particles. Journal of Biological Chemistry, 10;245(19):5129–5136.

Zelitch I, (2007). Properties of a new glyoxylate reductase from leaves. Biochemistry Journal 84(3):541–546.