The United Nations biennial report on world population predicts an increase from 7.2 billion in 2013 to 10.9 billion by 2100. This will result in ever greater demands for resources and food as well as pressure on the environment. Richard Smalley, co-recipient of the 1996 Nobel Prize in Chemistry, developed a list of the “top ten problems facing humanity over the next 50 years”. When these ‘wicked problems’ are ranked according to the degree to which solving a particular problem will facilitate solving others, the top 4 are energy, water, food and the environment.
Protein engineering and other nanoscale sciences possess remarkable potential to provide new environmentally friendly technologies to address a number of specific challenges in these 4 broad categories. For example, the use of enzymes in industrial applications diminishes energy requirements and the need for harsh chemicals and solvents and increases process yield. Medical applications are similarly diverse and include the development of novel therapeutics.
Enzyme engineering and biotechnology
Our lab is well positioned for the transdisciplinary approach required for this research. Our distinct blend of expertise spans protein biochemistry, biomedical, microbiology and molecular biology, encompassing a range of systems. The resulting diversity of techniques employed provides a strong training environment for graduate and undergraduate students.
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, enzyme kinetics, stopped-flow, circular dichroism spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy and calorimetry, to name a few.
This research has important applications, including:
1. Homocysteine metabolism in health and disease
Cellular metabolism, the sum of the reactions of a cell’s various enzymes, must be finely balanced to ensure the health of an organism. Ordinarily the cells of our body are able to achieve this remarkable feat. However, when one of the many different enzymes that a cell needs to maintain homeostasis is lacking or not functioning properly, the result can be visualized as a multi-car pileup on a busy highway. The effect is invariably not limited to the cellular level and is demonstrated in clinical symptoms, the severity of which depend on the specific enzyme affected and the degree of its impairment.
The enzyme cystathionine β-synthase (CBS) occupies a central position in cellular metabolism and plays a key role in regulating the flux of metabolites in several important pathways (e.g. as a traffic light at a major urban intersection). It is also the primary source of hydrogen sulfide in the central nervous system. Hydrogen sulfide is a neuromodulator and signaling molecule at low concentrations, but it is also a potent toxin and therefore its production in living cells must be strictly regulated. Diseases associated with imbalances in CBS function include Down syndrome and homocystinuria, which result from excess or deficiency of CBS, respectively, and therefore require distinct therapeutic strategies. The more than 100 distinct homocystinuria-associated mutations of the gene encoding CBS further complicates the development of therapeutics as each produces a distinct version of the disease such that effective treatment requires an approach adjusted to match the specific patient. Ongoing research in our lab aims to elucidate the complex regulatory mechanisms of this enzyme, as well as the structure-function relationships that underlie the various disease-associated mutations of this enzyme, with the goal of providing the framework on which the development of novel therapeutics and treatments will be based.
2. Enzyme engineering and biotechnology
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. Enzymes, as nature’s version of nanotechnology, hold great potential for biotechnology and are currently employed in many applications (e.g. pulp and paper, textiles, agri-food, etc). Advantages of enzymes, compared to the traditional chemical methods employed by many industries, include cost savings, increased yield and low environmental impact. This is because enzymes have evolved to function under mild conditions (i.e. inside the cells of our body) and to be very selective, both for the specific substrate and reaction catalyzed. However, while the range of reactions for which enzymes are available is impressive, they are limited to those required by biological organisms. The increasing array of molecular biology tools available has enabled the field of protein engineering to develop in recent years, providing us with the opportunity to expand the range of biotechnological, biomedical and industrial applications for enzymes by changing their properties, including stability under specific conditions (e.g. temperature, shelf life) and substrate and/or reaction specificity.
The versatile pyridoxal 5′-phosphate (PLP) cofactor catalyzes an array of reactions and specificity is provided by the protein scaffold of the enzyme to which it is bound. We are investigating the structure-function relationships that underlie substrate, inhibitor and reaction specificity and the mechanism whereby PLP-dependent enzymes regulate the chemistry of the cofactor. The enzymes of the microbial (including Escherichia coli, Saccharomyces cerevisiae and Trichomonas vaginalis) transsulfuration pathways, which interconvert the sulfur-containing amino acids cysteine and homocysteine, provide an ideal model system for this purpose. Although they share common structural features, they catalyze distinct side-chain rearrangements of similar amino-acid substrates. Our goal is to apply the knowledge we gain in the exploration of this system in the development of tailored catalysts for biotechnological or industrial applications and to facilitate the development of enzyme-specific inhibitors as antimicrobial compounds because the bacterial pathway is not present in mammals and therefore presents possibilities for the development of antiinfectives.