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Rubisco enzymes have been identified in most autotrophic species. So far four structural forms of Ru- bisco or Rubisco like enzymes have been identified in prokaryotes (bacteria, archea) and eukaryotes (algae, higher plants) [115].

Rubisco enzymes are always multimers and can contain a large (~52 kDa) and sometimes a small subunit (~15 kDa). Besides the sequence of the large subunit, the presence or absence of the small subunit is one classifying feature of Rubisco sequences and structures. Of the four types of Rubisco structures characterised so far, only form I contains a small subunit, whereas the oligomerization of the large subunits to at least dimers is retained in all forms. Figure 2.6 shows an overview of the different forms of Rubisco that are described in the following paragraphs. Only form I-III Rubiscos catalyse the name giving affixation of CO2or O2to RuBP [115].

Figure 2.6: Representative structures of the different forms of Rubisco.

All forms are comprised of antiparallel dimers of large subunits. Form I forms tetramers of dimers and is the only form that requires the presence of the small subunit. Form II and Form IV are structurally highly similar and form a single antiparallel dimer of large subunits. Form III Rubisco forms tetramers or pentamers of dimers, shown here is the pentameric form. Large subunits are shown in in green and yellow, small subunits are shown in blue. All structures are shown in a ribbon representation and are drawn to scale. PDB accession numbers are indicated in brackets under the species name 1RCX [116], 5RUB [117], 1GEH [118] , 1YKW [119].

2.4.1.1 Form I

Form I Rubiscos are further subdivided into Form I A-D. Form I A and B are found in cyanobacteria, pro- teobacteria, green algae and plants and thus are commonly referred to as green-type Rubiscos, whereas

form I C and D are found mostly in proteobacteria and rhodophytes and thus are called red-type Rubiscos [115] [120]. While the branching of form I enzymes probably occurred before the first endosmybiotic event, phylogenetical analysis suggest that branching into the green and red lineage occurred after the branching in form I-IV [115].

Figure 2.7: Structure of the large and small subunit of Rubisco.

A: Structure of the antiparallel dimer of form I Rubisco large subunits (from Nicotiana tabacum) shown in a ribbon representation colored in blue and brown shades respectively. N-terminal mixedβ-sheet domains are shown in darker shades, C-terminalα/β-barrel domains are shown in lighter shades. Large and small subunits forming the hexadecameric L8S8holoenzyme are not displayed. The structure of a tight binding inhibitor 2-carboxy-arabinitol-

bisphosphate (CABP) bound to the active sites is shown in a ball and sticks representation with carbon atoms colored in green, oxygen atoms colored in red and phosphate atoms colored in orange. PDB accession number 4RUB [121]. B: Superposition of small subunit structures from different species. All structures are shown as ribbon representations. Structures are color coded according to their origin: Nicotiana tabacum green 4RUB [121], Synechococcus elongatus PCC 6301 orange 1RBL [122], Chlamydomonas reinhardtii blue 1GK8 [123], Galdieria partita red 1BWV [124]. Variable loops are named according to their adjacentβ-sheets. Not the variability of the AB loop, as well as the unique insertion of the additionalβ-sheets EF at the C-terminus of the small subunit of the red-type Rubisco from Galdieria partita.

All Form I enzymes are hexadecameric complexes consisting of a tetramer of antiparallel dimers of the large subunits capped by four small subunits at the top and bottom, respectively. This gives the complex a square prism ’422’ symmetry. The active site is formed in all bona fide Rubiscos (Form I-III) by a dimerization of the large subunits mediated by mostly charged interactions. The carboxy-terminal α/β-

barrel domain of one large subunit forms the active site with the amino-terminal mixedβ-sheet domain of the dimerization partner [120] [125] (see Figure 2.7 A). The active site opens and closes during catalysis, mostly by movement of a loop (loop 6) and ordering of the extreme C-terminus of the enzyme [126].

While the structure of the large subunit is highly conserved in all Rubiscos, the structure of the small subunit is more diverse with the common core consisting of a four stranded anti-parallel β-sheet. The loop between strands A and B together with the extreme C-terminus show the highest sequence and structural divergence [127] (see Figure 2.7 B). Although the small subunit does not have a direct role in catalysis, chimeric Rubisco complexes show different kinetic properties e.g. in the specificity for the substrate CO2[127] [128].

2.4.1.2 Form II

Form II Rubisco consists of a single, antiparallel dimer of large subunits, forming two active sites by dimerization much like in form I Rubiscos. Found in Proteobacteria and some Dynoflagellates, form II Rubisco was used as a model protein for the catalysis and folding/assembly of Rubisco for decades, mainly because the structure of the enzyme of Rhodospirillum rubrum was solved already in 1986 [129] (later refined at higher resolution [117]) and the enzyme could be expressed heterologously.

2.4.1.3 Form III

Form III Rubisco sequences were first identified in large genomic scale sequencing studies in archaea [130]. They tend to form dimers similar to the form II enzyme or larger oligomeric states, for example tetramers or pentamers of dimers [131] [118] (PDB ID: 2CXE, 2CWX, 2D69 unpublished).

2.4.1.4 Form IV

Form IV Rubisco are not bona fide Rubiscos and show substitution in key active site residues involved in CO2 fixation. They have been suggested to bind different substrates for enolase/lyase type reactions [132] of e.g. the methionine salvage pathway. The Rubisco-like proteins identified so far assemble into antiparallel dimers of large subunits and are structurally closely related to the form II enzymes [133].