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Enzyme Evolution Reveals Earth’s Inhospitable Past

Photo: Ruth Arcus

Stromatolites found in Kalbarri National Park, Western Australia. Photo: Ruth Arcus

By Joanne Hobbs & Vic Arcus

The reconstruction of a one-billion-year-old enzyme paints a picture of a hot and hostile past.

The difficulty with studying extinct organisms is exactly that – they’re extinct. It is even more difficult to study the first microbes on Earth because, unlike the dinosaurs, they were too small to leave their imprints in the rocks as fossils.

Some microbes did build rocky structures that still exist today. These microbial fossils, such as the stromatolites found in parts of Western Australia, provide invaluable clues about the microorganisms that lived on Earth billions of years ago. But there are so many things that they can’t tell us about, such as the environments these organisms inhabited and how they adapted to changes in their environment.

We also face this same obstacle when we want to study the evolutionary history of these ancient organisms. How can we study the distant past and ancient evolutionary events when they’ve left so few clues for us to decipher?

We have recently used information contained in the DNA of modern microbes to reconstruct a one-billion-year-old enzyme. We then made this enzyme in the laboratory and studied its biochemical properties.

Our results revealed that the microbial host of this enzyme lived in a hot and inhospitable environment one billion years ago.

Choosing an Enzyme to Reconstruct
In order to reconstruct an enzyme from an extinct organism, you need to begin with its contemporary descendents and work backwards. We chose to work with modern Bacillus bacteria. Members of this genus are found almost everywhere in nature and, of particular interest to us, different species live at very diverse temperatures – from 0°C in Antarctica to 80°C in hot springs.

In the past 30 years, there has been heated debate about whether ancient organisms lived in hot or cold environment. Were ancient organisms thermophilic, and thrived above 60°C, or did they live at ambient temperatures or even in cold environments? This was a question we were keen to test using reconstructed enzymes from ancestral Bacillus species.

The particular enzyme we chose to reconstruct is called LeuB. This enzyme is a core component of the metabolism of most bacteria, and catalyses the third step in the biosynthetic pathway that produces the essential amino acid leucine. In the absence of a functional LeuB enzyme, a bacterium must rely on leucine obtained from its environment for survival.

LeuB was a challenging enzyme to reconstruct as it is structurally very complex and contains a number of binding sites. Errors during the reconstruction process could potentially disrupt these binding sites or the overall structure of the enzyme, which would result in an inactive enzyme.

Our ability to measure the activity of LeuB acted as an important check for the accuracy of our ancient sequence inference.

The Reconstruction Process
Once we had chosen an enzyme, we began the reconstruction process by comparing the amino acid sequences of LeuB from 19 modern Bacillus species. By aligning these sequences with each other, we could clearly see which amino acids had been conserved throughout evolution and which had been subject to change. We also compared the DNA sequences from these species that encode LeuB and this provided further information about how these amino acid changes had arisen from mutations in the DNA.

With this information we were able to build a tree of contemporary Bacillus species, which shows the relatedness of the different species to each other. The contemporary species represent leaves on the tree; the lengths of the branches indicate the number of amino acid differences between species; and the branch points within the tree represent ancestral species that no longer exist on Earth.

Using a molecular clock for evolution, we calculated that the last common ancestor of all modern Bacillus species would have lived approximately one billion years ago, when the Earth was mainly inhabited by microorganisms and the first, simple multicellular organisms were only just beginning to emerge.

Using our Bacillus tree and the LeuB sequence alignments, we were able to infer the amino acid sequence of LeuB at each of the ancestral branch points in the tree. This type of inference uses a computer algorithm that takes into consideration the amino acids found at each position in the enzyme in the modern species and the amount of evolutionary time that has passed.

We decided to reconstruct four ancestral LeuB enzymes in the laboratory and characterise them biochemically. These enzymes are positioned at different time points throughout the Bacillus tree and, as such, allow us to trace the evolution of this enzyme from one billion years ago up to the present day.

In order to characterise the biochemical and biophysical properties of an ancestral enzyme, we have to physically produce quantities of it in the laboratory. This is a three-step process.

First, a DNA sequence encoding the ancestral enzyme has to be chemically synthesised. Next, this DNA is introduced into the bacterium E. coli, which is tricked into producing the ancestral enzyme. Finally, once the ancestral enzyme has been produced, we can open the E. coli cells and extract the enzyme for biochemical testing.

The Billion-Year-Old Enzyme
When we first tested the activity of our billion-year-old enzyme, named ANC4, we noticed several incredible things. This enzyme differs from its closest modern descendent by 88 amino acids (a 24% difference) yet it is still fully functional, which suggests that our ancestral inference was accurate. We also found that ANC4 works seven times faster than the fastest modern enzyme.

ANC4 is also extremely stable at high temperatures. Enzymes are only active if they maintain their three-dimensional structure, and high temperatures cause enzymes to unfold, or melt. However, ANC4 works optimally at 70°C, has a high physical melting temperature and unfolds incredibly slowly.

We have also visualised the three-dimensional structure of ANC4 using a technique called X-ray crystallography, and we can see the amino acids that make this enzyme so stable.

So what does this tell us about the host of this enzyme and the environment it lived in? It has been shown previously, with modern enzymes, that the optimal working temperature and melting temperature of an enzyme are correlated with the growth temperature of its host. Therefore, we can infer that the host of ANC4 lived in a hot and inhospitable environment that necessitated such a hardy enzyme.

Given ANC4’s ability to survive above 60°C we were keen to investigate how this thermophilic billion-year-old Bacillus ancestor evolved to give rise to modern Bacillus species that inhabit environments ranging in temperature from 0°C to 80°C. To do this, we used the other three LeuB enzymes that we had reconstructed from younger ancestors of the Bacillus.

By characterising these enzymes in the same way as we did for ANC4, we were able to see that over time there has been a fluctuation in the temperature adaptation of Bacillus species. One billion years ago, the Bacillus began as a thermophilic organism but then around 850 million years ago it gradually adapted to much cooler temperatures, and then returned to thermophily again approximately 670 million years ago.

This finding goes against the accepted theory that thermophily has only evolved once in history.

The World One Billion Years Ago
One billion years ago, the Earth was in the middle of the geological period known as the Proterozoic eon. This was an eventful time on Earth, with tectonic collisions causing the formation of a supercontinent, the oxygenation of the atmosphere and the occurrence of several glaciations. These are the types of environmental changes that our one-billion-year-old enzyme and its host would have had to endure.

The stability and speed of this enzyme suggests that it had to function under extreme conditions, but the fact that it then evolved into less stable and slower enzymes suggests that it was not the wonder enzyme it appears. The regulation of an enzyme is a fine balance between meeting the needs of the cell and not wasting precious energy. It is also important for a cell to be able to break down enzymes when they are not required so it can build new ones. Perhaps, as the host entered a less extreme environment, this fast and long-lasting enzyme no longer provided a selective advantage.

Today, enzymes are used in hundreds of biotechnological applications, including food and drink production, medical diagnostics and the pharmaceutical industry. If other ancestral enzymes display similar properties to those of our billion-year-old enzyme, these enzymes may be of use to us in the modern world.

Box: Ancestral Sequence Reconstruction
Enzymes are proteins that act as biological catalysts. They are made up of chains of amino acids that fold up into a specific three-dimensional structure. Amino acid sequences are encoded by sections of DNA, or genes, within an organism’s genome. Therefore, every protein inside an organism has an amino acid sequence and a corresponding DNA sequence.

In 1963, Pauling and Zuckerkandl proposed that one day scientists would be able to infer the amino acid sequences of ancient proteins based on the DNA and amino acid sequences of modern proteins. They also suggested that, using advanced molecular techniques, these ancient proteins would be reconstructed in the laboratory and studied. The work of Pauling and Zuckerkandl forms the basis of what modern molecular biologists have termed ancestral sequence reconstruction.

Enzymes and proteins are molecular machines essential to life, and their activities and biophysical properties are finely tuned to the needs of their host organism. For example, if an organism lives in the soil surrounding a volcano then its proteins need to be able to maintain their three-dimensional structure at temperatures above 60°C, and its enzymes need to be able to catalyse biological reactions efficiently at high temperatures.

However, while it is important for proteins and enzymes to maintain their functionality, they also need to have the ability to adapt and evolve to changes in the environment. This evolution comes about as a result of random mutation within the organism’s genome.

Mutations that provide an organism with a selective advantage will be maintained in the genome and passed on to future generations, whereas mutations that have a negative effect will be removed from the population, either by reversal of the mutation or the death of the mutated cell. It is this trail of mutations within an organism’s genome that allows us to track backwards the evolution of its proteins and enzymes, which ultimately can reveal some incredible features of ancestral organisms.

We have used ancestral sequence reconstruction to reconstruct a one-billion-year-old enzyme and followed its evolution up to the present day.

Joanne Hobbs is a postdoctoral research scientist and Vic Arcus is an Associate Professor in the Department of Biological Sciences, University of Waikato, New Zealand.