The bottom line of this research
is extremely important. It means that we
can now predict that we will produce useful oils and other molecular feedstocks
through the means of agriculture that can happily displace oil based
petrochemicals which we are certainly tiring of using.
There is plenty to do but the
first application is likely not too far off.
Been able to squeeze canola oil from the meal and to then split off a commercial
fraction that then goes to industrial applications is now a prospect to be
taken up in the decade ahead.
I expect that we will now succeed
in retiring the classic petroleum industry with technology such as this. This is an important step forward.
Scientists Solve Long-Standing Plant Biochemistry Mystery
ScienceDaily (Sep. 19, 2011) — Scientists at the U.S. Department
of Energy's (DOE) Brookhaven National Laboratory and collaborators at the
Karolinska Institute in Sweden have discovered how an enzyme "knows"
where to insert a double bond when desaturating plant fatty acids.
Understanding the mechanism -- which relies on a single amino acid far from the
enzyme's active site -- solves a 40-year mystery of how these enzymes exert
such location-specific control.
The work, published in theProceedings of the National Academy
"Plant fatty acids are an approximately $150-billion-dollar-a-year
market," said Brookhaven biochemist John Shanklin, lead author on the
paper. "Their properties, and therefore their potential uses and values,
are determined by the position of double bonds in the hydrocarbon chains that
make up their backbones. Thus the ability to control double bond positions
would enable us to make new designer fatty acids that would be useful as
industrial raw materials."
The enzymes responsible for double-bond placement, called desaturases,
remove hydrogen atoms and insert double bonds between adjacent carbon atoms at
specific locations on the hydrocarbon chains. But how one enzyme knows to
insert the double bond at one location while a different but closely related
enzyme inserts a double bond at a different site has been a mystery.
"Most enzymes recognize features in the molecules they act on that
are very close to the site where the enzyme's action takes place. But all the
carbon-hydrogen groups that make up fatty-acid backbones are very similar with
no distinguishing features -- it's like a greasy rope with nothing to hold
onto," said Shanklin.
In describing his group's long-standing quest to solve the desaturation
puzzle, Shanklin quotes Nobel laureate Konrad Bloch, who observed more than 40
years ago that such site-specific removal of hydrogen "would seem to
approach the limits of the discriminatory power of enzymes."
Shanklin and his collaborators approached the problem by studying two
genetically similar desaturases that act at different locations: a castor
desaturase that inserts a double bond between carbon atoms 9 and 10 in the
chain (a 'delta-9' desaturase); and an ivy desaturase that inserts a double
bond between carbon atoms 4 and 5 (delta-4). They reasoned that any differences
would be easy to spot in such extreme examples.
But early attempts to find a telltale explanation -- which included
detailed analyses of the two enzymes' atomic-level crystal structures -- turned
up few clues. "The crystal structures are almost identical," Shanklin
said.
The next step was to look at how the two enzymes bind to their
substrates -- fatty acid chains attached to a small carrier protein. First the
scientists analyzed the crystal structure of the castor desaturase bound to the
substrate. Then they used computer modeling to further explore how the carrier
protein "docked" with the enzyme.
"Results of the computational docking model exactly matched that
of the real crystal structure, which allows carbon atoms 9 and 10 to be
positioned right at the enzyme's active site," Shanklin said.
Next the scientists modeled how the carrier protein docked with the ivy
desaturase. This time it docked in a different orientation that positioned
carbon atoms 4 and 5 at the desaturation active site. "So the docking
model predicted a different orientation that exactly accounted for the
specificity," Shanklin said.
To identify exactly what was responsible for the difference in binding,
the scientists then looked at the amino acid sequence -- the series of 360
building blocks that makes up each enzyme. They identified amino acid locations
that differ between delta-9 and delta-4 desaturases, and focused on those locations
that would be able to interact with the substrate, based on their positions in
the structural models.
The scientists identified one position, far from the active site, where
the computer model indicated that switching a single amino acid would change
the orientation of the bound fatty acid with respect to the active site. Could
this distant amino-acid location remotely control the site of double bond
placement?
To test this hypothesis, the scientists engineered a new desaturase,
swapping out the aspartic acid normally found at that location in the delta-9
castor desaturase for the lysine found in the delta-4 ivy desaturase. The
result: an enzyme that was castor-like in every way, except that it now seemed
able to desaturate the fatty acid at the delta-4 carbon location. "It's
quite remarkable to see that changing just one amino acid could have such a
striking effect," Shanklin said.
The computational modeling helped explain why: It showed that the
negatively charged aspartic acid in the castor desaturase ordinarily repels a
negatively charged region on the carrier protein, which leads to a binding
orientation that favors delta-9 desaturation; substitution with positively
charged lysine results in attraction between the desaturase and carrier protein,
leading to an orientation that favors delta-4 desaturation.
Understanding this mechanism led Ed Whittle, a research associate in
Shanklin's lab, to add a second positive charge to the castor desaturase in an
attempt to further strengthen the attraction. The result was a nearly complete
switch in the castor enzyme from delta-9 to delta-4 desaturation, adding
compelling support for the remote control hypothesis.
"I really admire Ed's persistence and insight in taking what was
already a striking result and pushing it even further to completely change the
way this enzyme functions," Shanklin said.
"It's very rewarding to have finally solved this mystery, which
would not have been possible without a team effort drawing on our diverse
expertise in biochemistry, genetics, computational modeling, and x-ray
crystallography.
"Using what we've now learned, I am optimistic we can redesign
enzymes to achieve new desirable specificities to produce novel fatty acids in
plants. These novel fatty acids would be a renewable resource to replace raw
materials now derived from petroleum for making industrial products like
plastics," Shanklin said.
This work was funded by the DOE Office of Science. Additional
collaborators include: Jodie Guy, Martin Moche, and Ylva Lindqvist of the
Karolinska Institute, and Johan Lengqvist, now at AstraZeneca R&D in Sweden
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