Mankind triumphed in a recent 'competition' against nature when scientists succeeded in creating a new type of enzyme for a reaction for which no naturally occurring enzyme has evolved. This achievement opens the door to the development of a variety of potential applications in medicine and industry.

Enzymes are, without a doubt, a valuable model for understanding the intricate works of nature. These molecular machines – which without them, life would not exist – are responsible for initiating chemical reactions within the body. Millions of years of natural selection have fine-tuned the activity of such enzymes, allowing chemical reactions to take place millions of times faster. In order to create artificial enzymes, a comprehensive understanding of the structure of natural enzymes, their mode of action, as well as advanced protein engineering techniques is needed. A team of scientists from the University of Washington, Seattle, and the Weizmann Institute of Science, Israel, made a crucial breakthrough toward this endeavor. Their findings have recently been published in the scientific journal Nature.

Enzymes are biological catalysts that are made from a string of amino acids, which fold into specific three-dimensional protein structures. The scientists’ aim was to create an enzyme for a specific chemical reaction whereby a proton (a positively charged hydrogen atom) is removed from carbon – a highly demanding reaction and rate-determining step in numerous processes for which no enzymes currently exist, but which would be beneficial in helping to speed up the reaction. During the first heat of the 'competition,' the research team designed the 'heart' of the enzymatic machine – the active site – where the chemical reactions take place. 

The second heat of the competition was to design the backbone of the enzyme, i.e., to determine the sequence of the 200 amino acids that make up the structure of the protein. This was no easy feat seeing as there is an infinite number of ways to arrange 20 different types of amino acids into strings of 200. But in practice, only a limited number of possibilities are available as the sequence of amino acids determines the structure of the enzyme, which in turn, determines its specific activity. Prof. David Baker of the University of Washington, Seattle, used novel computational methodologies to scan tens of thousands of sequence possibilities, identifying about 60 computationally designed enzymes that had the potential to carry out the intended activity. Of these 60 sequences tested, eight advanced to the next 'round' having showed biological activity. Of these remaining eight, three sequences got through to the 'final stage,' which proved to be the most active. Drs. Orly Dym and Shira Albeck of the Weizmann Institute’s Structural Biology Department solved the structure of one of the final contestants, and confirmed that the enzymes created were almost identical to the predicted computational design.

But the efficiency of the new enzymes could not compare to that of naturally-occurring enzymes that have evolved over millions of years. This is where 'mankind' was on the verge of losing the competition to nature, until Prof. Dan Tawfik and research student Olga Khersonsky of the Weizmann Institute’s Biological Chemistry Department stepped in, whereby they developed a method allowing the synthetic enzymes to undergo 'evolution in a test tube' that mimics natural evolution. Their method is based on repeated rounds of random mutations followed by scanning the mutant enzymes to find the ones who showed the most improvement in efficiency.

These enzymes then underwent further rounds of mutation and screening. Results show that it takes only seven rounds of evolution in a test tube to improve the enzymes’ efficiency 200-fold compared with the efficiency of the computer-designed template, resulting in a million-fold increase in reaction rates compared with those that take place in the absence of an enzyme.

The scientists found that the mutations occurring in the area surrounding the enzyme’s active site caused minor structural changes, which in turn, resulted in an increased chemical reaction rate. These mutations therefore seem to correct shortcomings in the computational design, by shedding light on what might be lacking in the original designs. Other mutations increased the flexibility of the enzymes, which helped to increase the speed of substrate release from the active site.
 
'Reproducing the breathtaking performances of natural enzymes is a daunting task, but the combination of computational design and molecular in vitro evolution opens up new horizons in the creation of synthetic enzymes,' says Tawfik. 'Thanks to this research, we have gained a better understanding of the structure of enzymes as well as their mode of action. This, in turn, will allow us to design and create enzymes that nature itself had not ‘thought’ of, which could be used in various processes, such as neutralizing poisons, developing medicines, as well as for many further potential applications.'  

Prof. Dan Tawfik's research is supported by the J & R Center for Scientific Research; the Jack Wolgin Prize for Scientific Excellence; Mr. and Mrs. Yossie Hollander, Israel; Mr. Rowland Schaefer, New York, NY; and the estate of Fannie Sherr, New York, NY.  Prof.  Tawfik is the incumbent of the Elaine Blond Career Development Chair.

The Weizmann Institute of Science in Rehovot, Israel, is one of the world's top-ranking multidisciplinary research institutions. Noted for its wide-ranging exploration of the natural and exact sciences, the Institute is home to 2,600 scientists, students, technicians and supporting staff. Institute research efforts include the search for new ways of fighting disease and hunger, examining leading questions in mathematics and computer science, probing the physics of matter and the universe, creating novel materials and developing new strategies for protecting the environment.

Weizmann Institute news releases are posted on the World Wide Web at
http://wis-wander.weizmann.ac.il/, and are also available at http://www.eurekalert.org/.

But in the last few years it has become clear that the very processes that generate mutations, if they take place at a relatively low frequency, can actually protect us from cancer. How does the body know how to keep these processes in check, making sure they don’t rocket out of control, causing a sharp rise in our cancer risk? A preliminary answer to this question has come out of research carried out by Prof. Zvi Livneh and research student Sharon Avkin, along with research student Leanne Toube and Dr. Ziv Sevilya of the Biological Chemistry Department, and Prof. Moshe Oren of the Molecular Cell Biology Department, along with two American colleagues. The results of their study appeared recently in the scientific journal Molecular Cell.

The instruments of DNA copying (which takes place prior to cell division) are members of a family of enzymes called DNA polymerase. DNA polymerase travels along one strand of the double stranded molecule, reading each bit of genetic material and copying as it goes along, to create new DNA that will be passed on to the daughter cell at cell division. This enzyme can be a stickler for accuracy – if it runs into damage from radiation or exposure to harmful substances on the DNA strand, it can stop in its tracks, unable to continue copying. A stoppage of this sort spells death for the cell. But not all damage to DNA is critical and, to avoid the wholesale death of cells, a second type of DNA polymerase, one that is more 'careless' and can improvise when it hits a snag, evolved in the cell. 'Error-prone DNA repair,' as it’s called, is based on a compromise: The cell lives, but at the price of allowing genetic mutations to be carried over in cell division.

The body’s solution to minimizing mutations is to have no fewer than ten different 'careless' enzymes. Although this may seem paradoxical – intuitively, more careless enzymes should mean more mutations – each of these enzymes is tailored to deal with certain specifics types of DNA damage. This specialization is what keeps the level of mutation, and thus the cancer risk, low.  But the existence of this variety of specialist enzymes implies precise regulation of the system – otherwise copying by the careless enzymes might get out of control and lead to an unhealthy proliferation of mutations.

Prof. Livneh and his team recently discovered a security mechanism that prevents such proliferation of mutations. This mechanism allows the right enzyme to go to work at the right time, and only when it’s needed. The main components in this system are the proteins p53 and p21. p53, named 'molecule of the year' several years ago by Science, is well known for its central role in reining in cancer processes in the cell. In this case, the proteins seem to act as supervisors, taming the careless enzymes and keeping them in careful check. The scientists’ research showed that if the functioning of p53 or its relative, p21, is harmed, the activities of the careless enzymes can go into overdrive, leading to more mutations.

The actual mechanism works with a sort of molecular clamp that holds the DNA copying enzyme onto the strand of DNA. When the enzyme encounters DNA damage, a small molecule called ubiquitin attaches to the clamp. The ubiquitin, in this case, serves to anchor replacement DNA polymerase molecules – careless ones – to the clamp. p53 enters the picture when it is alerted to the damage and causes p21 to be created. The p21 then acts as a sort of facilitator, helping to fasten the proper ubiquitin in place and clearing stalled DNA polymerase out of the way so its replacement can get to work. Thus, these two proteins manage to help the body’s cells maintain a crucial balance, allowing them to divide and multiply while keeping the mutation rate, and therefore the cancer risk, to a minimum.

Prof. Zvi Livneh’s research is supported by the M.D. Moross Institute for Cancer Research; the Dr. Josef Cohn Minerva Center for Biomembrane Research; the J & R Center for Scientific Research; the Levine Institute of Applied Science; the Flight Attendant Medical Research Institute; and the Israel Science Foundation. Prof. Livneh is the incumbent of the Maxwell Ellis Professorial Chair in Biomedical Research.

The Weizmann Institute of Science in Rehovot, Israel, is one of the world's top-ranking multidisciplinary research institutions. Noted for its wide-ranging exploration of the natural and exact sciences, the Institute is home to 2,500 scientists, students, technicians and supporting staff. Institute research efforts include the search for new ways of fighting disease and hunger, examining leading questions in mathematics and computer science, probing the physics of matter and the universe, creating novel materials and developing new strategies for protecting the environment.

Weizmann Institute news releases are posted on the World Wide Web at
http://wis-wander.weizmann.ac.il/, and are also available at http://www.eurekalert.org/.

The team found that the disease is caused by a mutation in the gene known as Calsequestrin 2 (CASQ2), which plays a vital role in the contraction and relaxation of the heart. The mutation impairs the ability of the CASQ2 protein to attract and release calcium ions upon demand.

Published in the American Journal of Human Genetics, the study was performed by doctoral student Hadas Lahat, her advisors, Prof. Michael Eldar, Chief of the Heart Institute at the Sheba Medical Center and Dr. Elon Pras of the Danek Gartner Institute of Human Genetics, Sheba Medical Center, and Dr. Avidan, Dr. Tsviya Olender, Dr. Edna Ben-Asher, and Prof. Doron Lancet of the Weizmann Institute's Department of Molecular Genetics.

The study began when an eight-year-old girl in the tribe fainted following physical exertion, and was admitted to the Rambam Medical Center. Her two younger brothers suffered from the same symptoms, and two other siblings had previously died of the disease. While the hospital physicians believed that the children's symptoms were due to tachycardia, they were unable to identify the precise cause. The family then turned to Prof. Michael Eldar, Chief of the Heart Institute at the Sheba Medical Center, who diagnosed the children as suffering from PVT. During his meeting with them, Eldar learned that several other families in the village had a similar medical history.

The girl's family belongs to a Bedouin community in northern Israel, thought to be the descendants of three brothers who had settled there over 200 years ago. The nature of the disease and the common Bedouin custom of familial intermarriage alerted researchers to possible genetic involvement. Hypothesizing that one of these brothers had harbored a genetic mutation, Lahat, of the Danek Gartner Institute of Human Genetics, visited the community and tested the families. It appeared she was on the right track. In seven families alone, thirteen children were identified as having PVT and were given appropriate medication. Nine untreated children in these families had earlier died from this condition.

But the researchers had yet to identify the exact gene responsible for PVT from a possible 80 candidate genes. At the time the Human Genome Project was far from complete and information was available for only half of the 80 suspect genes. Lahat and her supervisor, Dr. Elon Pras, decided to approach Weizmann Institute scientists for help.

At the Institute Drs. Olender and Avidan initially had little success, yet as the Human Genome Project generated new information at an increasing speed, the researchers fortuitously came upon a newly mapped gene, Calsequestrin 2 (CASQ2), just as they were losing hope. The CASQ2 protein appeared as a good candidate because it serves as a calcium ion reservoir in heart muscle cells. By binding, holding, and releasing calcium ions, the CASQ2 protein could thus play a key role in the contraction and relaxation of heart muscles. A second clue came from an unexpected source. Only four months earlier a different research team had found that a mutation in another gene, known as RYR2, also causes a form of PVT. Furthermore, RYR2 was found to belong to the same cellular pathway as CASQ2.

The researchers soon found that the children suffering from PVT had a mutation in their CASQ2 gene, and were able to pinpoint how things had earlier gone wrong in those that had died. They discovered that the mutation was surprisingly small -- characterized by only a single base change, from G to C, in one of the DNA's nucleotides. Nevertheless this change causes the body to produce the amino acid histidine instead of aspartic acid, which impairs the CASQ2 protein's ability to attract and release calcium ions.

'The protein carries a very strong negative charge, thus binding a large number of positively charged calcium ions and releasing them when necessary,' says Avidan. 'Unfortunately, in the mutated CASQ2 protein, the overall negative charge is smaller, since the single base change replaces the aspartic acid which is negatively charged -- with the positively charged histidine. This most likely damages the protein's ability to attract calcium ions, leading to heart failure.'

Other scientists collaborating in this study are Etgar Levy-Nissenbaum and Dr. Boleslaw Goldman of the Danek Gartner Institute of Human Genetics and Dr. Asad Khoury and Dr. Avraham Lorber of the Rambam Medical Center.

Donor support: The Crown Human Genome Center and the Israel Science Foundation Grant

The Weizmann Institute of Science is a major center of scientific research Israel. Its 2,500 scientists, students and support staff are engaged in more than 1,000 research projects across the spectrum of contemporary science.