Successful improvement of the catalytic activity of photosynthetic CO2
fixing enzyme Rubisco

Date:
September 15, 2020
Source:
Kobe University
Summary:
A research group have succeeded in greatly increasing the catalytic
activity of Rubisco, the enzyme which fixes carbon from carbon
dioxide in plant photosynthesis. The research team also hypothesized
the mechanism which determines the enzyme's catalytic activity. In
the future, it is hoped that increasing the photosynthetic ability
of agricultural crops will lead to improved yields.



FULL STORY ==========================================================================
A research group consisting of Associate Professor FUKAYAMA Hiroshi
(Kobe University, Graduate School of Agricultural Science) and Professor MATSUMURA Hiroyoshi (Ritsumeikan University) et al. have succeeded in
greatly increasing the catalytic activity of Rubisco (*1), the enzyme
which fixes carbon from CO2 in plant photosynthesis. The research team
also hypothesized the mechanism which determines the catalytic activity
of Rubisco, based on structural analysis of the proteins.


==========================================================================
In the future, it is hoped that increasing the photosynthetic ability
of agricultural crops will lead to increased yields.

These results were published in the international scientific journal
Molecular Plant on August 31.

Main Points
* Photosynthesis determines a plant's growth rate. The low activity
of the
enzyme Rubisco, which is the catalyst for the reaction that turns
CO2 into organic carbon, limits the rate of photosynthesis.

* Rubisco consists of two types of protein; large subunits (RbcL)
and small
subunits (RbcS). RbcS is an important factor for determining the
speed of the catalyst.

* A hybrid Rubisco consisting of rice RbcL and sorghum RbcS
demonstrated a
catalytic rate that was approximately 2 times higher than that of
rice Rubisco. This is believed to be the first time in the world
that such a large increase in Rubisco activity has been achieved.

* The 102 amino acid found inside RbcS is isoleucine in rice and
leucine in
sorghum. Analysis of the protein structures indicated the
possibility that the difference in amino acid type could affect
the catalytic activity.

* It is hoped that the method to improve photosynthesis demonstrated
in
this study could be applied to many other crops which, like rice,
have low Rubisco activity such as wheat, soybean and potato.

Research Background Growth speed in plants is mainly determined by photosynthetic ability. Thus improving photosynthesis in agricultural
crops can increase their yield. In photosynthesis, Rubisco is an enzyme
that acts as the initial catalyst for the reaction which turns CO2
into organic carbon. However, Rubisco has two major drawbacks which
limit photosynthesis: its catalytic activity is very low, and it can be inhibited by O2 (ie. Rubisco can mistakenly fix to O2 molecules instead
of CO2 molecules, creating a toxic compound that needs to be recycled
by the plant).



========================================================================== Rubisco's catalytic activity varies depending on the type of plant. Most
major crops, such as rice, wheat and soybean are C3 plants that use
regular photosynthesis. C4 plants, such as corn and sugarcane, on
the other hand, have acquired a mechanism to concentrate CO2 (the C4 photosynthetic pathway).

The catalytic rate is low in C3 plants, whereas in C4 plants it
tends to be high. Rubisco with high catalytic activity tends to be
inhibited easily by oxygen, therefore it cannot function effectively in atmospheric conditions where there is a low concentration of CO2 if the
plant doesn't have a CO2- concentrating mechanism. However, as the amount
of atmospheric CO2 is continuing to increase, it is believed that if C3
plants had the same highly active type of Rubisco as C4 plants then this
could be utilized to improve photosynthetic ability.

Research Findings: Rubisco is made up of two types of protein- large
subunits (RbcL) and small subunits (RbcS). The sequence of the amino acids
in RbcS varies greatly between species. This team has been focusing on conducting research into RbcS. They genetically modified rice (a C3 plant)
by transferring RbcS from the C4 plant sorghum, successfully increasing
the catalytic rate of rice Rubisco 1.5 times.

This rice with sorghum RbcS inserted (SS line), produced a chimera form
of Rubisco from both sorghum RbcS and rice RbcS. Next, the rice RbcS
gene was knocked out in the sorghum RbcS incorporated rice plants using CRISPR/Cas9 gene editing.

In this CSS line (sorghum RbcS transferred/rice RbcS knocked out), the
rice RbcS was completely replaced by sorghum RbcS, producing hybrid
Rubisco. This approximately doubled the catalytic rate to that which
is equivalent to C4 plants. Although many researchers have been able to
improve Rubisco's catalytic characteristics, there have been no examples
of such a large increase being achieved. Furthermore, CSS line plants demonstrated a higher photosynthetic rate than unmodified (wild type)
rice under high CO2 conditions, even though the amount of Rubisco in
their leaves was over 30% less.



========================================================================== Subsequently, the researchers conducted x-ray crystallography (*2)
in order to illuminate the mechanism by which sorghum RbcS increases
Rubisco's catalytic activity. RbcL is present in Rubisco's catalytic
site. Near this catalytic site, there is a structure called RbcS
bC. The 102 amino acid found in bC is isoleucine in rice and leucine
in sorghum. Leucine has smaller molecules than isoleucine. Therefore,
it is thought that in sorghum RbcS the gaps between amino acid molecules
become bigger, making the reaction site more pliable and thus increasing catalytic activity. Although further research is necessary to prove this,
it is believed to be a previously unproposed ground-breaking theory for
Rubisco research.

Conclusion The CSS line produced in this study demonstrated high
photosynthetic ability, however crop yield was not improved. Hopefully,
it will be possible to vastly improve plant growth and productivity
through appropriate control of Rubisco levels.

The current research used the C3 plant rice, however it is vital to
consider the applications of this methodology and investigate whether
or not the same strategy can be used to increase Rubisco's catalytic
activity in other major crops, such as wheat, soybean and potato.

It is thought that the 102 amino acid is an important determinant of the catalytic activity. Further research is being carried out to investigate
this; for example by replacing only the amino acid at the 102 site with
another amino acid and producing Rubisco.

Glossary 1. Rubisco (Ribulose-1,5-biphosphate carboxylase/oxygenase):
In photosynthesis, the enzyme Rubisco is the catalyst for the
initial reaction that fixes CO2. It is not only the catalyst for the photosynthesis reaction (carboxylase reaction) but also serves as the
catalyst for the oxygenase reaction where O2 is the substrate. Plants accumulate large amounts of Rubisco in their leaves to compensate for the
very slow rate of the catalyst. Around half of the soluble proteins in
rice plant leaves are Rubisco and it is known to be the most prevalent
protein on Earth.

2. X-ray crystallography: A method to determine the spatial coordinates of
the atoms that make up a crystal by analyzing the diffraction obtained
from x- raying a single crystal. This is one way of determining the
structural arrangement of proteins. It is necessary to produce protein crystals.


========================================================================== Story Source: Materials provided by Kobe_University. Note: Content may
be edited for style and length.


========================================================================== Journal Reference:
1. Hiroyoshi Matsumura, Keita Shiomi, Akito Yamamoto, Yuri Taketani,
Noriyuki Kobayashi, Takuya Yoshizawa, Shun-ichi Tanaka, Hiroki
Yoshikawa, Masaki Endo, Hiroshi Fukayama. Hybrid Rubisco
with Complete Replacement of Rice Rubisco Small Subunits by
Sorghum Counterparts Confers C4-Plant- like High Catalytic
Activity. Molecular Plant, 2020; DOI: 10.1016/ j.molp.2020.08.012 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/09/200915090123.htm

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