Natural Protein, 27th of March 2003
- Its 3-D structure -
|We have already
introduced the research on protein structure and function
to maintain our life activities several times. Revealing the
sophisticated 3-D structure of protein would enable us to
determine how each protein performs its function. KEK investigates
the 3-D structure of protein by using synchrotron radiation
produced by the accelerator. Today, we will introduce the
research on natural protein, by using the synchrotron radiation
research facility of KEK, which binds one carbon to the other
and promotes the reaction that synthesizes a cyclic compound
of carbon that is tortoise shell-like.
Reaction that produces tortoise shell of carbon
Our bodies are mostly made of carbon compounds. Figure 1 shows
the important reaction that produces the carbon compound.
This reaction is promoted spontaneously when dienophile, which
is a compound with one double bond, and diene, with two double
bonds, approach each other, and cyclohexene, which is the
tortoise shell-like carbon compound, is produced. The discovery
of this reaction, which is rational and does not need intense
condition such as heating, has led to the dramatic development
of organic synthetic chemistry and the revolution in the synthetic
industry. Diels and Alder who discovered this reaction were
awarded the 1950 Nobel Prize in chemistry. This is called
the Diels-Alder reaction, one of the most well-known reactions
Natural protein that promotes Diels-Alder reaction
It has long been thought that the Diels-Alder reaction may
be important in not only synthetic chemistry but also in nature.
The Diels-Alder reaction was predicted to be present in natural
synthetic reactions since there are many carbon compounds
with the tortoise shell-like skeleton among the compounds
created by certain microorganisms. Some of these reactions
are shown in Figure 2. The parts that correspond to diene
and dienophile, or the parts of the skeletons produced from
these reactions in the synthesized compounds are colored.
The occurrence of these reactions in nature indicates the
presence of the proteins that act to keep stable "transiting
conditions," such as the blue-circled part in Figure
1. The only roles of these proteins are to fix the direction
to make the reaction of diene and dienophile easier. Now,
how do the proteins fix the direction of these carbon compounds?
In the research that we introduce here, we revealed a sophisticated
protein strategy from its 3-D structure through examining
the 3-D structure of macrophomate synthetase, a natural protein
that promotes the reaction shown in Figure 2-c.
Multistep catalyst macrophomate synthetase
A certain fungus that forms leaf spots on Commelina communis
produces abundantly 2-pyrone or macrophomate, which are carbon
compounds with tortoise shell-like skeletons and have a physiological
function as phytotoxins. In fact, 2-pyrone is transformed
to macrophomate by a single enzyme, macrophomate synthetase.
The reaction promoted by this enzyme is shown in Figure 3.
The red-circled part demonstrates the production of a new
carbon skeleton given by oxaloacetic acid, another substrate,
in the middle of the reaction. You can see in Figure 3 that
the second step of this complicated, multistep reaction pathway
is the Diels-Alder reaction.
3-D structure of macrophomate synthase
Figure 4 shows the 3-D structure of macrophomate synthase
clarified at the synchrotron radiation research facility in
KEK. The structure is comprised of six molecules, a hexamer.
The hexamer structure is necessary for this protein to act.
The hexamer has six active sites containing Magnesium ion
(Mg2+). The important result of this 3-D structural study
is the elucidation of the structure of a complex that contains
the compounds corresponding to substrates diene or dienophile
in its protein. Figure 5 shows how the substrates gain access
to each other when the reaction occurs in an active site.
Mg2+ or the side chain of the protein tightly fixes the two
The enzyme that catalyzes the Diels-Alder reaction is artificially
made using an antibody, which is a life activity that recognizes
foreign bodies. This enzyme is called abzyme. Compared to
the abzyme with highest efficiency among these artificial
ones, macrophomate synthase, of which the structure has been
revealed this time, has much higher catalytic efficiency.
This is because of the extremely rational design of naturally
occurring protein. Other highly sophisticated design ideas
that we have not revealed yet might be hidden in natural protein
structures. This research not only helps to unlock the secrets
of the natural world but also enables the development of sophisticated
natural devices within our life, such as making new effective
This research was conducted by Professor Isao Tanaka's group
of Hokkaido University and published in Nature, 13 March 2003.
Diels-Alder reaction. When diene and dienophile gain access
to each other, effectively from beneath and from above, two
new carbon-carbon bonds are formed. The figure shows only
skeletons, although diene and dienophile with various functional
groups are practically used.
Among naturally occurring compounds, the 3 compounds solanapyrone,
lovastatin, and macrophomate, are predicted to pass through
the Diels-Alder reaction in their synthetic process and identify
the presence of proteins. Proteins that catalyze them are
SPS (Solanapyrone synthetase), LNKS (lovastatin nonaketide
synthetase), and MPS (macrophomate synthetase), respectively.
This time, MPS was used to analyze the 3-D structure(c). All
3 enzymes are multi-step catalysts that catalyze not only
the Diels-Alder reaction but also even the preliminary step.
magnification (23 KB)
Macrophomate synthetase-catalyzed pathway. In the first step,
the enzyme acts as rapid oxaloacetate decarboxylase. The Diels-Alder
reaction then occurs as the second step between pyruvate enolate,
the production of the first step, and 2-pyrone. The reactant
of the second step is converted to the end product macrophomate
through the decarboxylation and the dehydration as the third
step. Since the environment around the protein active site
has been elucidated through this study, it has become possible
to consider specifically this complex pathway.
magnification (26 KB)
General structure of macrophomate synthetase. Each 6 molecules
are color-corded. Such hexamer structure is necessary for
the protein to act. The red balls indicate magnesium ions
at active sites.
magnification (94 KB)
The substrates, 2-pyrone (diene, orange) and pyruvic acid
(dienophile, dark pink) at active sites. These 2 substrates
are firmly fixed through the interaction (light blue dotted
line) between magnesium (green) and protein side-chain. The
pink dot indicates the carbon-carbon bond that is subsequently
magnification (38 KB)
For further information
-» Laboratory of Structural Bio-Macromolecular Sciences
2, Hokkaido University (Professor Tanaka's laboratory)
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