Sue Aitken

Research

Enzymology and Sulfur Amino Acid Metabolism

My research focuses on exploring the mechanism and regulation of the enzymes involved in sulfur amino acid regulation in plants, humans, and microorganisms. We use an array of methods including molecular biology, enzyme kinetics, and biophysical spectroscopy. Techniques applied include: PCR and real-time PCR, site-directed mutagenesis, steady state and presteady state kinetics, stopped-flow, circular dichroism spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, isothermal titration calorimetry, and analytical ultracentrifugation, to name a few. This research has important agricultural, medical, and biotech applications, including:

1. Biosynthesis of methionine in plants
   Legumes tend to be deficient in the essential amino acid methionine and are therefore an incomplete source of protein for human consumption. The development of new varieties of legume crops with increased levels of methionine would be of economic benefit not only to Canada, a major producer and exporter of these crops, but on a larger scale as well, as legume crops are a staple in many countries. Sulfur amino acid biosynthesis and accumulation is under complex metabolic control. The increased understanding, both of the enzymes involved and of their transcriptional and post-translational regulation, will lead to the development of crop plants with improved nutritional characteristics.
2. Phytoremediation of soil contaminated by heavy metals
   Phytoremediation is an environmentally-friendly tool for the removal of toxic compounds, such as heavy metals, from contaminated soils. It involves the use of plants to extract metal contaminants from soil. Plants that are effective in this role are referred to as hyperaccumulators. The thiol groups of sulfur-containing compounds, such as the amino acid cysteine, are known to be good coordinators of metal ions and likely play a role in their uptake by plants. Our research focuses on probing the distinct genetic features of hyperaccumulators, in particular those related to sulfur metabolsim. Long term goals include the generation of plant varieties that are well suited to phytoremediation.
3. Metabolism of homocysteine
   The medical applications of our research relate to homocysteine, an intermediate of methionine metabolism that has been recognized as an independent risk factor for heart disease. Homocysteine levels in the blood are usually in the low-micromolar range. However, even a moderate increase in the plasma concentration of homocysteine, resulting from a variety of genetic, dietary, environmental, and drug-induced conditions, is associated with an increased risk of arteriosclerosis in humans. According to the most recent (1999) statistics from Health Canada , cardiovascular disease (CVD) is the leading cause of death amongst Canadians, accounting for 36% of all deaths. Homocystinuria is an autosomal, recessive disease characterized by elevated plasma homocysteine levels. The most common cause of this disease in humans is deficiency of the enzyme cystathionine beta-synthase. Cystathionine beta-synthase may also be linked to Down syndrome. The elucidation of the complex and interlocking mechanisms of cystathionine beta-synthase regulation and the underlying mechanisms of homocystinuria-associated mutations will lead to the design of therapeutic treatments for those at risk for atherosclerosis and to prevent the clinical manifestations of homocystinuria and Down syndrome.
4. Biotech applications and enzyme engineering
   Most chemical reactions occur too slowly to support life. Enzymes are biological molecules, generally proteins, which catalyze chemical reactions under the physiological conditions of the living cell. Although the 20 amino acids found in proteins provide an array of chemical tools for catalysis, enzymes frequently require additional assistance from small molecules, referred to as cofactors, which may be metal ions or organic molecules. Enzymes catalyzing transformations of amino acids are generally dependent on pyridoxal 5’-phosphate (PLP). As this versatile cofactor catalyzes a variety of reactions, the reaction specificity is provided by the protein component of the enzyme. Our research is employs a number of approaches including site-directed mutagenesis, phylogenetic and structural analysis, and directed evolution to probe the mechanism of substrate and reaction specificity in PLP-dependent enzymes. Future goals and applications of this work include the engineering of enzymes with novel activities for biotechnological applications. .

Selected Publications

Aitken SM, Kirsch JF. The enzymology of cystathionine biosynthesis: strategies for the control of substrate and reaction specificity. Archives of Biochemistry and Biophysics 2005. 433(1):166-75.

Ouellet M, Aitken SM, English AM, Percival MD

Aromatic hydroxamic acids and hydrazides as inhibitors of the peroxidase activity of prostaglandin H2 synthase-2. Archives of Biochemistry and Biophysics 2004. 431(1):107-18.

Aitken SM, Kirsch JF. Role of active-site residues Thr81, Ser82, Thr85, Gln157, and Tyr158 in yeast cystathionine beta-synthase catalysis and reaction specificity.

Biochemistry. 2004. 43(7):1963-71.

Aitken SM, Kim DH, Kirsch JF. Escherichia coli cystathionine gamma-synthase does not obey ping-pong kinetics. Novel continuous assays for the elimination and substitution reactions. Biochemistry. 2003. 42(38):11297-306.

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