Strategy of 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 among chemists.

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 substrates.

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 catalysts.

This research was conducted by Professor Isao Tanaka's group of Hokkaido University and published in Nature, 13 March 2003.

Figure 1
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.
High magnification (13KB)





Figure 2
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.
High magnification (23 KB)





Figure 3
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.
High magnification (26 KB)




Figure 4
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.
High magnification (94 KB)





Figure 5
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 formed.
High magnification (38 KB)

For further information

-» Laboratory of Structural Bio-Macromolecular Sciences 2, Hokkaido University (Professor Tanaka's laboratory)
http://altair.sci.hokudai.ac.jp/g6/

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