Georgia Tech School of Biology associate professor Eric Gaucher and graduate student Zi-Ming Zhao discuss the evolutionary history of the thioredoxin gene family, which provides clues about early life on Earth. Credit: Gary Meek
new study reveals that a group of ancient enzymes adapted to substantial
changes in ocean temperature and acidity during the last four billion years,
providing evidence that life on Early Earth evolved from a much hotter, more
acidic environment to the cooler, less acidic global environment that exists
study found that a group of ancient enzymes known as thioredoxin were
chemically stable at temperatures up to 32 degrees Celsius (58 degrees
Fahrenheit) higher than their modern counterparts. The enzymes, which were
several billion years old, also showed increased activity at lower pH levels—which
correspond to greater acidity.
study shows that a group of ubiquitous proteins operated in a hot, acidic
environment during early life, which supports the view that the environment
progressively cooled and became more alkaline between four billion and 500
million years ago,” said Eric Gaucher, an associate professor in the School of Biology at the Georgia Institute of
study, which was published in an advance online edition of Nature Structural
& Molecular Biology, was conducted by an international team of
researchers from Georgia Tech, Columbia
Univ., and the Universidad de Granada
funding for this study was provided by two grants from the National Aeronautics
and Space Administration to Georgia Tech, a grant from the National Institutes
of Health to Columbia
Univ., and a grant from
the Spanish Ministry of Science and Innovation to the Universidad de Granada.
a technique called ancestral sequence reconstruction, Gaucher and Georgia Tech
biology graduate student Zi-Ming Zhao reconstructed seven ancient thioredoxin
enzymes from the three domains of life—archaea, bacteria, and eukaryote—that
date back between one and four billion years.
resurrect these enzymes, which are found in nearly all known modern organisms
and are essential for life in mammals, the researchers first constructed a
family tree of the more than 200 thioredoxin sequences available from the three
domains of life. Then they reconstructed the sequences of the ancestral
thioredoxin enzymes using statistical methods based on maximum likelihood.
Finally, they synthesized the genes that encoded these sequences, expressed the
ancient proteins in the cells of modern Escherichia
coli bacteria and then purified the proteins.
resurrecting proteins, we are able to gather valuable information about the
adaptation of extinct forms of life to climatic, ecological and physiological
alterations that cannot be uncovered through fossil record examinations,”
reconstructed enzymes from the Precambrian period—which ended about 542 million
years ago—were used to examine how environmental conditions, including pH and
temperature, affected the evolution of the enzymes and their chemical
Eric Gaucher, an associate professor in the Georgia Tech School of Biology, examines resurrected proteins from an evolved family of fluorescent proteins. Gaucher is using them to validate the tools he uses to resurrect ancient proteins. Credit: Gary Meek
the ancient origin of the reconstructed thioredoxin enzymes, with some of them
predating the buildup of atmospheric oxygen, we thought their catalytic
chemistry would be simple, but we found that thioredoxin enzymes use a complex
mixture of chemical mechanisms that increases their efficiency over the simpler
compounds that were available in early geochemistry,” said Julio
Fernández, a professor in the Department of Biological Sciences professor at
led a team that included Columbia
researchers Raul Perez-Jimenez, Jorge Alegre-Cebollada, and Sergi
Garcia-Manyes, and graduate student Pallav Kosuri in using an assay based on
single molecule force spectroscopy to measure the activity level of the
thioredoxin enzymes under different pH levels.
their experiments, the researchers used an atomic force microscope to pick up
and stretch an engineered protein in a solution containing thioredoxin. They
first applied a constant force to the protein, causing it to rapidly unfold and
expose its disulfide bonds to the thioredoxin enzymes. The rate at which a
thioredoxin enzyme snipped the disulfide bonds determined the enzyme’s level of
study results showed that the three oldest thioredoxin enzymes—those thought to
have inhabited Earth 4.2 to 3.5 billion years ago—were able to operate in lower
pH environments than the modern thioredoxin enzymes.
analysis indicates that ancient thioredoxin enzymes were well adapted to
function under acidic conditions and that they maintained their high level of
activity as they evolved in more alkaline environments,” said Fernández.
measure the temperature range in which the enzymes operated, professor Jose
Sanchez-Ruiz and graduate student Alvaro Inglés-Prieto from the Departamento de
Química-Física at the Universidad de Granada in Spain used a technique called
differential scanning calorimetry. This method measures the stability of
enzymes by heating the enzymes at a constant rate and measuring the heat change
associated with their unfolding.
researchers found that the ancient proteins were stable at temperatures up to
32 degrees Celsius higher than the modern thioredoxins. The experiments showed
that the enzymes exhibited higher temperature stability the older they were.
The results provide evidence that ancestral thioredoxins adapted to the cooling
trend of ancient oceans, as inferred from geological records.
results confirm that life has the remarkable ability to adapt to a wide range
of historical environmental conditions; and by extension, life will undoubtedly
adapt to future environmental changes, albeit at some cost to many
species,” said Gaucher.
study also showed that the experimental resurrection of ancient proteins
together with the sensitivity of single-molecule techniques can be a powerful
tool for understanding the origin and evolution of life on Earth.
researchers are currently using this strategy to assess other enzymes to get a
clearer picture of what life was like on Early Earth. They are also applying
these tools to the field of biotechnology, where enzymes play important roles
in many industrial processes.
functions and characteristics we observed in the ancestral enzymes show that
our techniques can be implemented to generate improved enzymes for a wide range
of applications,” added Perez-Jimenez.