Dr Christina Divne, from the Karolinska
Institutet, highlights her work on sugardegrading enzymes and the potential
possessed by these fascinating proteins
an important part of the decay process that
enables the fungus to utilize wood.
Could you tell us about the specific qualities
of the sugar-oxidising enzyme produced by
wood-rotting fungi, P2O, and what drew you
to study this fungi and enzyme?
Can you provide an overview of the aims and
focus of your research? What are the central
questions driving your project?
The scope of my research for the last twenty
years has been to understand how enzymes
work, and specifically enzymes that act on
sugars crucial for the fundamental chemistry of
wood decay. Sugar compounds have enormous
importance for all life forms, from simple
single-cell organisms, to land-living trees and
highly evolved vertebrates like humans. A few
examples of where sugars perform vital roles
include structural reinforcement of plants,
energy storage and supply, cellular signalling,
immune system development, as pathogenic
virulence factors in pathogenic microorganisms,
and in DNA/RNA. A new project on
carbohydrate-active enzymes that is currently
in the start-up phase concerns enzymes that
synthesise polysaccharides.
As in the case of CDH, this enzyme participates
in degradation of plant matter, but seems to
be more important for degradation of lignin
than cellulose. The fact that the most efficient
lignin-degrading fungi all produce P2O made it
particularly interesting to study in the context of
biomass recycling. In addition, redox chemistry
is very fascinating but highly complex, while
flavin chemistry is very intricate. People have
studied the mechanisms of flavoenzymes for a
very long time, but since the precise chemistry
is highly context-dependent, each enzyme
presents a new challenge. It turned out that
P2O was a particularly suitable enzyme to study
mechanistically, since it was possible to produce
crystal structures of the enzyme with several
types of bound sugars. This provided deepened
insight regarding fundamental processes such
as the high selectivity in oxidation position. I
was also very attracted to the many possible
biotechnological applications of the enzyme,
such as a wide repertoire of sugar intermediates,
from simple and easily available substrates,
intermediates that can be used to produce
valuable sugar compounds.
Further to this, what might the applications
of such an enzyme be if it were reproducible
on an industrial scale?
Some applications have already been
patented, such as the P2O-catalysed
production of fructose by Cetus in 1981.
Other examples include production of
tagatose, which is an artificial sweetener
with prebiotic properties, the antioxidant
isoascorbic acid, and the antibiotic
cortalcerone. There are also attractive
applications of P2O in the fields of biosensors
and biofuel cells.
DR CHRISTINA DIVNE
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More than a
sweet tooth
If we go back to CDH, this enzyme has
been successfully used in development
of biosensors for monitoring of blood
glucose levels in diabetic patients, work
that was pioneered by Prof. Lo Gorton at
Lund University. In addition, CDH can be
used for bioremediation and environmental
monitoring of environmentally hazardous
compounds, for instance, degradation of
polyacrylate and explosives such as TNT.
The sugar-oxidising enzymes with natural
functions in degradation of plant material
also have natural relevance to applications in
biomass processing, although less than the
hydrolytic enzymes (cellulases).
Are there any other aspects of your research
you would like to highlight here?
The new project concerning polysaccharidesynthesising enzymes is very exciting.
The fascinating aspect of some synthases
is the ability to polymerise very long
polysaccharides (thousands of sugar units),
for instance cellulose and hyaluronan,
inside the cell, and then translocate the
polysaccharide to the extracellular side.
The molecular mechanisms underlying
these processes are completely unknown
at present, and a challenge that I look very
much forward to.
Why are sugar-transforming enzymes’
role in living organisms so important to
understand in general?
Many biochemical reactions in living organisms,
most importantly metabolic processes, depend
on sugars, and enzymes that catalyse the
conversion of one type of sugar to another
are usually active in metabolic pathways.
Since all cells require energy, a general
basic understanding of metabolic pathways,
whether it applies to humans or wood-rotting
fungi, is important in order to understand the
very core of life maintenance. For example,
removing the gene coding for CDH (cellobiose
dehydrogenase) in the rot fungus Trametes
versicolor efficiently prevents the fungus from
invading and colonising birch wood, with
starvation as a result. In the case of pyranose
2-oxidase (P2O), the primary function is
believed to be in lignin degradation, which is
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DR CHRISTINA DIVNE
Deconstructing the
deconstructors
Sugar-degrading enzymes play an
essential role within nature, especially
in the decomposition of plants. A team
from the Karolinska Institutet have
been working to uncover their secrets,
paving the way for a range of cuttingedge applications for the 21st Century
CARBOHYDRATES, OR SUGARS, play a crucial
role in all living organisms. Forming long chains
known as polysaccharides, they are the basis for a
variety of compounds with a myriad of functions.
For example, in cellulose form they provide
structure to plants and protection to bacteria,
while in their basic glucose form they are used
as an energy source. They are also a means of
energy storage: as starch in plants and glycogen
in animals. Other polysaccharides are essential to
immune function, cellular communication and as
a component of DNA and RNA.
The formation, destruction and modification of
polysaccharides are almost exclusively performed
by enzymes. These remarkable proteins are
present in all known life forms, and are capable
of catalysing a whole host of reactions that would
otherwise take place too slowly to be useful.
Sugar-degrading enzymes are used by a huge
range of microorganisms, and have an essential
role in breaking down plant matter. As such, they
play a key role in the global carbon cycle itself,
without which most life on
Earth would cease to exist.
Today, scientists are working to
unravel the
secrets of such enzymes, which may have a host
of uses, from breaking down household or toxic
waste, to manufacturing new carbohydrate-based
materials, to biomedical applications.
STRUCTURE EQUALS FUNCTION
In order to understand how sugar-transforming
enzymes function within biological systems,
a team of post-doctorate researchers from
the Karolinska Institutet in Sweden have been
using macromolecular X-ray crystallography
(also known as single-crystal X-ray diffraction)
to determine the structure of some of these
extraordinary entities. Led by Dr Christina Divne,
a crystallographer and structural biologist with
over twenty years experience in the field, the
team have focused their attention on sugaroxidising enzymes (sugar oxidoreductases), in a
project that initiated in 1997.
Of the three sugar oxidoreductases
that Divne and her team
have
studied,
cellobiose
dehydrogenase (CDH) was
the first to be investigated
in detail. This enzyme is
produced by wood-rotting
fungi, and is critical for
these organisms to invade
and colonise wood. It is
truly versatile, being able
to degrade not only
cellulose, but also lignin
and other carbohydrate
polymers that are
constituents
of
certain types of wood.
Divne’s studies have
shown that it oxidises sugars
specifically at the first carbon atom
in the molecule, and is the only known
extracellular enzyme that contains both a
flavin and haem group, which allows for electron
transfer from the flavin to the haem group when
oxidising cellobiose to produce lactone. These
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INTERNATIONAL INNOVATION
electrons are then passed to an external electron
acceptor, opening up the possibilities of direct
electron transfer (DET) applications.
The second of the sugar-oxidising enzymes
that Divne has studied in depth is pyranose
2-oxidase (P2O). This fascinating molecule is also
produced by wood-rotting fungi, and oxidises
If it’s not possible to design a better
and less costly process, an enzyme
will not be attractive for industry
a range of simple sugars at the second carbon
in each molecule. Manufactured by the onlyknown organisms capable of fully converting
lignin into carbon dioxide and water, the team
have discovered that these proteins contain a
large, water-filled cavity, within which the sugar
oxidation occurs. The purpose of this cavity is
still undetermined, although Divne speculates
that it may either be to block the exit of toxic byproducts, or to prevent entry of unsuitable sugars.
The most recent of the sugar-oxidising enzymes
that the team has been studying within this project
is pyranose dehydrogenase.This enzyme (or group
of enzymes) is produced by litterdecomposing
fungi, and like CDH and P2O also contains a flavin
co-factor. However, unlike P2O, it cannot use
oxygen to reduce the flavin when oxidising sugars,
but instead uses a variety of quinone compounds.
Being capable of transforming all major sugar
components of hemicellulose, this remarkable
enzyme is able to perform oxidation at multiple
sites on the sugar molecule.
APPLYING THE SCIENCE
So far, the team has had great success in
determining the structure of the sugar
oxidoreductases and their relative function. This is
a vital step, since mechanisms such as the position
of oxidation on the sugar molecule dictate
what the product of oxidation will be. Using
this knowledge has allowed them to engineer
the molecules, either by increasing stability or
changing the specificity of the substrate. They
have already done this with CDH and P2O,
which are being lined up for a range of potential
uses. For example, together with collaborators
in Vienna and Lund, they plan to improve the
properties of CDH to produce a new generation
of biocompatible, implantable long-term glucose
sensors for diabetics. Existing uses include
biomass processing and degrading hazardous
chemicals, such as TNT and polyacrylate. The
pyranose-oxidising enzymes also have many
potential uses in producing biofuel cells and
biosensors, and already are under patent for food,
chemical and drug production. Divne is cautiously
optimistic about the possibilities: “An enzyme
may have a range of important uses, but if it’s not
possible to design a process that is better and less
costly than the existing process, the enzyme will
not be attractive for industry,” she stresses.
In addition to her work on the sugar-oxidising
enzymes, Divne also has another active
project, which she started in March 2010. This
research focuses on elucidating the molecular
mechanisms
of
polysaccharide-producing
enzymes (polysaccharide synthases). So far, little
is known about these proteins, and Divne has
many questions she wishes to answer. These,
however, will not be easy to come by, she is
keen to stress: “The new project on structurefunction studies of polysaccharide-synthesising
enzymes will be particularly challenging, since
the enzymes are large proteins embedded in the
plasma membrane, and clearly our approach
will need to be both innovative and persistent
to achieve the goal”. The potential of held by
these marvellous molecules is huge though,
ranging from new methods to fight bacterial
infections to production of new
nanomaterials for a myriad
of uses.
INTELLIGENCE
STRUCTURE-FUNCTION STUDIES ON
PYRANOSE-OXIDIZING ENZYMES
OBJECTIVES
Our objective is to understand the structure
and function of important carbohydrateactive enzymes in molecular detail. How do
these enzymes perform vital functions? - and
how can this knowledge be used to create
a more sustainable development such as
improved human health, and more efficient
use of natural resources?
KEY COLLABORATORS
Professor Dietmar Haltrich, BOKU (Austria)
Dr Roland Ludwig, BOKU (Austria)
Associate Professor Clemens Peterbauer,
BOKU (Austria)
FUNDING
Postdoctoral researchers Rosaria Gandini (pictured) and
Tien-Chye Tan provide essential expertise to the team
ESSENTIAL TEAMWORK
Divne’s work is highly dependent on her team
of expert researchers. This includes Tien-Chye
Tan, a molecular biologist from Singapore who
is also trained in structural biology, and Rosaria
Gandini, who is an expert in protein production
and characterisation techniques. The team enjoys
a fruitful long-term research collaboration with
Professor Dietmar Haltrich, Dr Roland Ludwig
and Associate Professor Clemens Peterbauer (all
from BOKU, Austria). Divne is clearly still excited
by this meeting of minds, despite her long history
in the field: “Interdisciplinary research offers
exciting new angles to scientific problems,” she
explains. “Working at the interface between
disciplines is both fascinating and mind provoking,
and modern structural biology offers an exciting
blend of biology, chemistry and physics, which is
something I find very attractive.”
The Swedish Research Council VR
The Swedish Research Council Formas
The Carl Tryggers Foundation for Scientific
Research
CONTACT
Christina Divne
Karolinska Institutet
Dept. of Medical Biochemistry and
Biophysics (MBB) Division of Biophysics
Scheelelaboratoriet Scheeles väg 2
SE-171 77 Stockholm, Sweden
E [email protected]
http://ki.se/ki/jsp/polopoly.
jsp?d=33608&l=sv
CHRISTINA DIVNE was awarded a PhD in
Molecular Biology by Uppsala University in
1994. After starting her own independent
research in 1997, she moved to the Royal
Institute of Technology in 2001, where she
has been active as associate professor and
senior lecturer. Since 2010, she is an associate
professor at Karolinska Institutet.
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