Folding and assembly of RuBisCO

To become biologically active, proteins have to acquire their
correct three-dimensional structure by folding, which is frequently
followed by assembly into oligomeric complexes. Although all
structure relevant information is contained in the amino acid
sequence of a polypeptide, numerous proteins require the assistance
of molecular chaperones which prevent the aggregation and promote
the efficient folding and/or assembly of newly-synthesized
proteins. The enzyme ribulose-1,5-bisphosphate
carboxylase/oxygenase (RuBisCO), which catalyzes carbon fixation in
the Calvin-Benson-Bassham cycle, requires chaperones in order to
acquire its active structure. In plants and cyanobacteria, RuBisCO
(type I) is a complex of approximately 550 kDa composed of eight
large (RbcL) and eight small (RbcS) subunits. Remarkably, despite
the high abundance and importance of this enzyme, the
characteristics and requirements for its folding and assembly
pathway are only partly understood. It is known that folding of
RbcL is accomplished by chaperonin and most likely supported by the
Hsp70 system, whereas recent findings indicate the additional need
of specific chaperones for assembly. Nevertheless, this knowledge
is incomplete, reflected by the fact that in vitro reconstitution
of hexadecameric RuBisCO or synthesis of functional plant RuBisCO
in E. coli has not been accomplished thus far. In this thesis,
attempts to reconstitute type I RuBisCO in vitro did not result in
production of active enzyme although a variety of reaction
conditions and additives as well as chaperones of different kind,
origin and combination were applied. The major obstacle for
reconstitution was found to be the incapability to produce RbcL8
cores competent to form RbcL8S8 holoenzyme. It could be shown that
the RbcL subunits interact properly with the chaperonin GroEL in
terms of binding, encapsulation and cycling. However, they are not
released from GroEL in an assembly-competent state, leading to the
conclusion that a yet undefined condition or (assembly) factor is
required to shift the reaction equilibrium from GroEL-bound RbcL to
properly folded and released RbcL assembling to RbcL8 and RbcL8S8,
respectively. Cyanobacterial RbcX was found to promote the
production of cynanobacterial RbcL8 core complexes downstream of
chaperonin-assisted RbcL folding, both in E. coli and in an in
vitro translation system. Structural and functional analysis
defined RbcX as a homodimeric, arc-shaped complex of approximately
30 kDa, which interacts with RbcL via two distinct but cooperating
binding regions. A central hydrophobic groove recognizes and binds
a specific motif in the exposed C-terminus of unassembled RbcL,
thereby preventing the latter from uncontrolled misassembly and
establishing further contacts with the polar peripheral surface of
RbcX. These interactions allow optimal positioning and
interconnection of the RbcL subunits, resulting in efficient
assembly of RbcL8 core complexes. As a result of the highly dynamic
RbcL-RbcX interaction, RbcS can displace RbcX from the
core-complexes to produce active RbcL8S8 holoenzyme.
Species-specific co-evolution of RbcX with RbcL and RbcS accounts
for limited interspecies exchangeability of RbcX and for
RbcX-supported or -dependent assembly modes, respectively. In
summary, this study helped to specify the problem causing
prevention of proper in vitro reconstitution of type I RuBisCO.
Moreover, the structural and mechanistic properties of RbcX were
analyzed, demonstrating its function as specific assembly chaperone
for cyanobacterial RuBisCO. Since the latter is very similar to
RuBisCO of higher plants, this work may not only augment the
general understanding of type I RuBisCO synthesis, but it might
also contribute to advancing the engineering of catalytically more
efficient crop plant RuBisCO both in heterologous systems and in
planta.