By Kangsan (Jason) Lee ’16, THURJ Staff
Nature has always been a valuable source of scientific innovation. In fact, a field of research called biomimicry seeks to understand natural biological mechanisms and devices and apply them to various situations. A fascinating modern example is the artificial leaf, designed by researchers at MIT, which consists of solar cells that imitate principles of photosynthesis to split water into hydrogen and oxygen using sunlight. Living organisms have evolved over billions of years, during which time nature has maximized the efficiency of tasks. Consequently, contemporary scientific ideas and concepts continue to draw inspiration from nature.
In the industrial world, chemical processes associated with producing pharmaceuticals and drugs often take place in diverse and extreme conditions. Raising the temperature and pressure during production can reduce the risk of contamination and significantly increase the rate of reactions, as collision rate is proportional to kinetic energy and collision frequency. Conducting drug synthesis in moderate conditions would result in very slow rates of desired product formation, since intermediary steps often have high kinetic energy barriers. Therefore, organometallic catalysts, which can function in extreme conditions, are utilized in drug synthesis reactions.
However, researchers have been recently bioprospecting, or using bacterial enzymes as alternatives to traditional organometallic catalysts. These enzymes are not only more environmentally friendly but also more efficient because organometallic catalysts often produce undesired waste productions, many of which are toxic.
The value of extremophile bacteria, for instance, is being increasingly recognized. Extremophile bacteria have truly adapted to survival in remarkable environments, including the hot springs of Yellowstone National Park, deep-sea hydrothermal vents near Papua New Guinea, and beneath sheets of ice in Siberia. Although most known enzymes exist in moderate conditions, researchers have been recently studying enzymes found in extremophile bacteria, which function optimally and resist chemical degradation in their environments, by analyzing their structures and assessing their roles in biological pathways.
In particular, decoding primary sequences and structures can reveal patterns and trends responsible for the stability of these enzymes. Perhaps with this knowledge, mutations can be introduced into known enzymes to nduce tolerance to extreme conditions. Researchers are particularly interested in studying proteases and lipases, which are naturally found in extremophile bacteria and break down proteins and lipids, respectively.
Understanding the mechanisms of proteases found in hyperthermophile bacteria are particularly useful, as proteases must bind tightly to specific and recognized peptide sequences in order to induce peptide bond hydrolysis or proteolysis. Thus, researchers have proposed the use of proteases as catalysts for the modification of peptide sequences.
A number of proteases that are tolerant to high temperatures have been identified in hyperthermophiles. Many of them seem to resemble subtilisin-like serine proteases, which contains the catalytic triad of serine, aspartate, and histidine in its active site. Prominent examples of these proteases include Tk-1689 and Tk-subtilisin, found in Thermococcus kodakaraensis. These proteases are able to withstand temperatures of over 100°C, but they function best between 80 and 90°C, respectively. In fact, compared to their protease counterparts found in mesophiles (organisms found in moderate conditions) these proteases display 30 times more activity at 80°C than at room temperature.
Researchers are also capitalizing on modern technology to discover enzymes with the specific properties that they desire. Rather than searching for rare, naturally occurring extremophile enzymes in geysers or ocean depths, researchers are able to form enzymes with desired characteristics through a process known as directed evolution. Essentially, researchers accelerate the process of evolution and natural selection in the lab by utilizing error-prone PCR to introduce mutations into genetic sequences that code for enzymes. Then, the enzymes translated from these altered sequences can be screened for enzymatic activity, such that enzymes that function effectively in extreme conditions can be randomly generated by this method. However, most of these mutations result in failure, and few succeed. In one instance, researchers genetically modified the mesophilic subtilisin E using random mutagenesis to produce a 12-mutation variant that was 470 times more active than the wild-type enzyme.
In addition to proteases, lipases are important to study because they have the ability to hydrolyze substrates containing hydroxyls, esters, and amines, which are present in many biomolecules used in drug synthesis. Furthermore, lipases can catalyze transesterification reactions that involve the exchange of R groups, which are critical for designing molecules with desired bonds and consequent properties.
Researchers have discovered Candida antarctica lipase B (CALB), which functions in temperatures around 70°C. Through error-prone PCR, they discovered a mutant with a much lower dissociation constant; in fact, the mutant had a half-life of around 220 minutes, a drastic improvement from the original wild type, which had a half-life of only 8 minutes.
In the aforementioned enzymes, researchers have identified the presence of conserved structural motifs via various methods, including infrared spectroscopy, x-ray crystallography, and nuclear magnetic resonance. Enzymes must be structurally stable and able to maintain an active conformation, while simultaneously remaining flexible enough to allow substrates and cofactors to bind in the active and allosteric sites. Certain peptide sequences are thus evolutionarily favored, as their resultant enzymes are more effective in accomplishing their functions. In extreme conditions, the enzymes must first be stable and resist denaturation before they can perform their functions. Therefore, enzymes are often fairly rigid, as high temperatures promote high levels of protein movement and, consequently, unfolding and denaturation. Therefore, various features of extremophile enzymes promote structural stability. Perhaps an in-depth understanding of these structural motifs will one day allow the designing of primary sequences that can generate enzymes with specific geometries and charge distribution, resulting in enzymes with desired capabilities.
A commonly observed motif is the abundant formation of disulfide bridges. Analyzing the favorability of these bridges, we can understand that they increase the kinetic stability of enzymes by increasing the activation energy barrier of unfolding. In more intricate examples, disulfide bridges are located even in the same subunit, allowing for the formation of peptide rings, which ultimately organize and catenate into complex connected networks, which is thermodynamically favorable.
In addition, extremophile enzymes feature more extensively utilize hydrophobic residues. Some studies suggest that the hydrophobic residues allow more efficient and denser protein packing in the hydrophobic interior, reducing contact with water and thus lowering the entropic losses in protein folding. Furthermore, this strong hydrophobic packing allows less surface area for interface with water, reducing the probability of water performing a nucleophilic attack on the wrong side chain group. Also, structural stability is enhanced by aromatic side chains, which can stack given correct orientation. Comparison between the wild type isocitrate dehydrogenase, found in Thermatoga maritima, and mutant type, featuring mutations in the extensive aromatic cluster of the hydrophobic interior of the wild type enzyme, shows that the wild type has greater structural stability, indicating the importance of aromatic stacking interactions in stability.
This emerging research shows great promise, as discovering enzymes from extremophile bacteria or genetically engineering known enzymes will be greatly beneficial to the chemical processes associated with developing and producing pharmaceuticals. Biological enzymes can be more effective and environmentally friendly than organometallic catalysts currently in use. Investing in the discovery and genetic modification of useful extremophile enzymes will result in pharmaceuticals being produced at a reduced cost. Ultimately, this will contribute to increased availability of pharmaceuticals to the public.