click to enlarge Structural biologist Subramaniam Eswaramoorthy stands at the NSLS X-ray beamline used to reveal the atomic structure of the bioremediation enzyme. The liquid nitrogen in the foreground keeps the crystallized enzyme cool, protecting it from the heat of the X-rays that would otherwise damage the crystal.

Some remarkable types of bacteria have proven themselves capable
of “consuming” toxic pollutants, organically diminishing environmental impact
in a process called bioremediation. Enzymes within these bacteria can
effectively alter the molecular structure of dangerous chemicals, but the
underlying mechanisms and keys to future advances often remain unknown.

Now, scientists at the U.S. Department of Energy’s (DOE) Brookhaven
National Laboratory have revealed a possible explanation for the superior
function of one pollution-degrading enzyme. Using X-ray diffraction techniques
at Brookhaven’s National Synchrotron Light Source (NSLS), they probed the
structure of an enzyme engineered at Stanford
University that had been
identified as particularly effective in dealing with a carcinogenic pollutant
known as hexavalent chromate. They discovered that this mutated enzyme might
owe its high performance to its unique structure of intermolecular bonds. The research,
published in PLoS ONE, offers insights
into the future engineering of bacteria for pollutant bioremediation.

“The enzyme’s structure surprised us,” said Brookhaven
structural biologist Subramaniam Eswaramoorthy, lead author on the paper along
with Sebastien Poulain of Stanford
University. “Typically,
you expect an enzyme’s so-called active site, which interacts directly with
hexavalent chromate, to determine reaction levels. But we found that the unique
intermolecular hydrogen bonds within this mutant enzyme, called ChrR, are what
most likely make it such a strong performer.”

click to enlarge The active site of the mutant ChrR enzyme, identified as particularly effective in the bioremediation of carcinogenic pollutants, can be seen here as a stick-model within the larger protein structure. The increased volume of the cavity may make the entry and subsequent alteration of toxic chromate easier than in the “wild” enzyme.

Hexavalent chromate, also known as Chromate VI, is a human
carcinogenic pollutant that occurs frequently as a byproduct of industrial
processes. Its ability to contaminate other substances, such as water supplies,
raises environmental concerns. An appropriate enzyme, however, can neutralize
and effectively inhibit toxicity by stripping the compound of its electrons.
While a few enzymes can perform this function, most fall into a vicious and
unproductive cycle.

“Most enzymes pull only one electron from Chromate VI, turning
it into a Chromate V intermediate,” Eswaramoorthy said. “But then that second
compound pulls an electron from other reactions and transforms itself right
back into Chromate VI. At that point the initial reaction begins again, and
this toxic pollutant just bounces back and forth between the two forms. This is
not a solution.”

The new mutant enzyme that is the subject of the current study,
which was isolated from the bacteria Esxcherichia coli, acts differently: It
uniquely converts Chromate VI into benign Chromate III in just one reaction,
leapfrogging the intermediate Chromate V. The mechanism is still not entirely
understood, but could be a simultaneous two-electron transfer. That action’s
demonstrated efficacy in bioremediation motivated the investigation into the
bacterial enzyme’s atomic structure.

Scientists recorded the patterns produced as intense X-rays from
NSLS bounced off the atoms in a sample of crystallized ChrR, a process known as
diffraction. This revealed details at the single angstrom level, spanning
distances of just one or two atoms. The protein structure deduced from these
diffraction images showed that the enzyme ChrR is a tetramer, or a biological
unit formed by four distinct protein molecules binding together.

click to enlarge This model of the highly effective form of the bioremediation enzyme ChrR shows the intermolecular interactions and unique multi-atom hydrogen bonds (dotted lines) that may be the key to understanding the enzyme’s efficacy in reducing the toxicity of some dangerous pollutants.

Critically, this promising enzyme features unexpected
intermolecular interactions. In the unmutated or “wild” enzyme, three protein
bonds are supported by a single atom; but in the mutant just tested, these
bonds are distributed across several atoms. That multi-atom reinforcement
grants greater stability to the enzyme—consider the difference between a camera
balanced on a tripod and another perched precariously on a single pole. The
distinct structure may also offer a larger space for toxic pollutants to enter
the protein active site and allow bioremediation reactions to occur.

“We’re talking about building a better, more stable machine,”
Eswaramoorthy said. “When substrates such as Chromate VI come and go, this
strongly bonded enzyme does the job more effectively. We believe that the
three-bond structure makes the difference.”

Bioremediation of dangerous pollutants such as hexavalent
chromate remains experimental, but the new research helps push this kind of
bacterial enzyme closer to real-world neutralization of such carcinogens. The
team also noted that this enzyme has potential uses in some kinds of cancer
treatment, for example, by fine-tuning the activation of drugs in targeted
chemotherapy.

Scientists in Stanford
University’s Department
of Microbiology and Immunology plan to continue to test the efficacy of various
forms of the enzyme for use in bioremediation. Brookhaven scientists will then
probe the atomic structures, looking for correlations between form and
function.

Brookhaven National Laboratory