Bringing silicon to life via directed evolution of Cytochrome C

Nature is diverse, but silicon-carbon bonds have not been observerd in earth’s biology.

Silicon is one of the most abundant elements on earth, yet nature’s biology completely neglected it when forming our carbon-based life. In a time when our biggest innovations happen in a silicon based universe, researchers are trying to understand if silicon instead of carbon could also have been used by bioactive molecules.

In a groundbreaking paper recently featured in the journal Science, scientists from the California Institute of Technology in Pasadena investigated if proteins could help with the synthesis of carbon-silicon bonds; a chemical liaison not found in nature.

Traditionally, organic chemistry relies a a variety of tricks to synthesize designer-molecules; for example chiral reagents or rare-earth element based catalysts need to be used in multiple-step reactions with many sideproducts and low yield of final product. For the addition of a carbon bond to a silicane (silicon based compound), organic chemistry would rely on rhodium or iridium catalysts, as well as halogenated solvents and low temperatures. Even then, turnover rates and yields would be poor.

Because of their ability to accelerate chemical reactions and exquisite specificity and selectivity, enzymes are increasingly sought-after to complement or replace chemical synthesis methods. -Kan S.B.J et al., Science, 2016

Jennifer Kan and her colleagues reasoned that a certain class of proteins called “heme proteins”, which utilize iron to insert a carbon bond between nitrogen-hydrogen or sulfur-hydrogen bonds, might be the ideal proteins to investigate for their silicon-hydrogen bond insertion potential.

The catalyzing element in heme protein is iron, which is abundantly available in living beings but thought incapable of catalyzing silicon-carbon bond formation. After all, in nature, we do not observe silicon-carbon bonds at all.

One of the most important aspects of scientific inquiry is experiment; just because we have never observed something does not mean that it does not exist. So Jennifer Kan and colleagues prepared an assay where they would use a silicane and a carbon-donor (Me-EDA) and spike in “heme-bound”-iron and later different members of the broad heme-family of proteins.

The addition of heme-bound iron or heme proteins to the reaction mix was able to catalyze silicon-carbon bound formation

Their speculation had been right, addition of heme proteins to the reaction mix would enable a small fraction of precursors to form silicon-carbon bonds, compared to no bonds observed in reactions without heme.

However, another big issue in synthetic biology are so-called enantiomers; chemical compounds identical in their makeup but mirrored; like twins. Unfortunately, for chemical enantiomers, we often have a Dr. Jakyl and Mr. Hyde situation, where one twin is an active beneficial drug, and the other one is poisonous compound. One sadly famous example of an enatiomer gone bad is the case of Thalidomide, an over-the-counter drug sold in Germany in the 60’s against morning sickness for pregnant women. It was proclaimed a “wonder drug” for insomnia, coughs, colds and headaches. Unfortunately, its evil look-alike enantiomer twin causes severe birth defects, which resulted in thousands of malformed childbirths and 40% child lethality. Since then, more strict regulations for “enantio-purity” have been installed by governments and have to be upheld by all synthetic chemists and biologists.

So ensuring enantio-purity is a big concern for any synthetic reaction, and Jennifer Kan and colleagues spend a considerable amount of time finding a heme protein that catalyzed the right enantiomer; they settled for Cytochrome C from Rhodothermus marinus [Rma cyt c], a Gram-negative, thermohalophilic bacterium from submarine hot springs in Iceland. Thanks again, diverse mother earth.

The crystal structure of wild type [Rma cyt c] protein allowed them to investigate the steric conditions in the catalytic center (also called binding pocket) of that protein and literally take a look on the interior of the protein. Like for any good designer (interior or other), form follows function, and by generating space where needed or introducing barriers where necessary, the researchers could boosts the function of the catalytic center towards silicon-carbon reactions.

Introducing mutations in the binding pocket of an enzyme changes its catalytic capacities. Similar to renovating an old room for a new function, space needs to modified.

Neatly enough, this “interior design” can not only improve catalytic turnover, but also improve on enantiomer selectivity. Biologists call this process of iteratively improving a protein “directed evolution”, but the reality of it is just testing some room-layout ideas by introducing different amino acids (“mutations”) into the coding sequences of the protein and assessing which changes improved the wanted reaction compared to which ones did not.

While this evolutionary path culminated in a catalyst that is able to channel most reactants through carbon-silicon bond formation (in fact the authors already were able to produce a plethora of different silica-carbon formations), true science always opens up more or deeper questions than it answered in the first place.

In a last amazing experiment, the authors asked whether organosilicon products could be produced by bacteria that are equipped with this “updated” form of cytochrome c. Biosynthesis is a whole different kind of world, because living things do not like weird temperatures, molecules, extreme pH-values and a whole other array of classical chemist’s tools to get the job done. And surprisingly, [Rma cyt c]-equipped bacteria could produce organosilicates with high yield and specificity in this whole-cell biological setting.

Which begs the question:

Why did nature never make use of this, since the capabilities were always only one mutated protein away?

In our vast universe, could there be a “natural” biology based on silicon?

These in vitro and in vivo examples of carbon–silicon bond formation using an enzyme and Earth-abundant iron affirm the notion that nature’s protein repertoire is highly evolvable and poised for adaptation: With only a few mutations, existing proteins can be repurposed to efficiently forge chemical bonds not found in biology and grant access to areas of chemical space that living systems have not explored. -Kan S.B.J et al., Science, 2016

Evolution got us so far, but maybe it’s time we humans finally take the initative and deliberatly explore spaces where we know no living thing has set foot ever before.

We did it once with the moon. We can do it again with synthetic biology.

Source: Kan S.B.J et al., Science, 2016

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