Rubisco: The Enzyme that Keeps on Giving
2007; Cell Press; Volume: 129; Issue: 6 Linguagem: Inglês
10.1016/j.cell.2007.06.002
ISSN1097-4172
Autores Tópico(s)Algal biology and biofuel production
ResumoIn cyanobacteria, the RbcX protein enhances the production of Rubisco, the multisubunit enzyme that catalyzes the first step of carbon dioxide fixation in most autotrophic organisms. In this issue of Cell, Saschenbrecker et al., 2007Saschenbrecker S. Bracher A. Rao K.V. Rao B.V. Hartl F.U. Hayer-Hartl M. Cell. 2007; (this issue)PubMed Google Scholar report that RbcX acts as a specific assembly chaperone that mobilizes the large subunits of Rubisco to a specific oligomeric core that can then combine with the small subunits of Rubisco to form the functional holoenzyme. In cyanobacteria, the RbcX protein enhances the production of Rubisco, the multisubunit enzyme that catalyzes the first step of carbon dioxide fixation in most autotrophic organisms. In this issue of Cell, Saschenbrecker et al., 2007Saschenbrecker S. Bracher A. Rao K.V. Rao B.V. Hartl F.U. Hayer-Hartl M. Cell. 2007; (this issue)PubMed Google Scholar report that RbcX acts as a specific assembly chaperone that mobilizes the large subunits of Rubisco to a specific oligomeric core that can then combine with the small subunits of Rubisco to form the functional holoenzyme. The most abundant enzyme in nature is Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), which catalyzes the first step of carbon dioxide fixation in the majority of photosynthetic and chemoautotrophic organisms. In eukaryotes, the catalytically competent Rubisco holoenzyme (form I) is fully active only when eight large (L) subunits and eight small (S) subunits form an (L2)4(S4)2 hexadecamer. Since its identification nearly 50 years ago, Rubisco has frequently emerged at the leading edge of important issues in biochemistry, cell and molecular biology, and structural biology. In particular, analysis of the assembly of the Rubisco holoenzyme in chloroplasts led to the discovery of molecular chaperones (Barraclough and Ellis, 1980Barraclough R. Ellis R.J. Biochim. Biophys. Acta. 1980; 608: 18-31Google Scholar). Subsequently, the GroE heat-shock proteins were found to act as molecular chaperones (Goloubinoff et al., 1989Goloubinoff P. Gatenby A.A. Lorimer G.H. Nature. 1989; 337: 44-47Crossref PubMed Scopus (505) Google Scholar) for the products of the L (rbcL) and S (rbcS) genes allowing their assembly into the fully active recombinant Rubisco of cyanobacteria (Gatenby et al., 1985Gatenby A.A. van der Vies S.M. Bradley D. Nature. 1985; 314: 617-620Crossref Scopus (59) Google Scholar, Tabita and Small, 1985Tabita F.R. Small C.S. Proc. Natl. Acad. Sci. USA. 1985; 82: 6100-6103Crossref PubMed Scopus (54) Google Scholar). Further studies using the simpler Rubisco from the purple nonsulfur bacterium Rhodospirillum rubrum, which consists of a homodimer of L subunits (form II), showed that GroEL and GroES facilitate reconstitution of the native form of the enzyme from unfolded monomers in vitro. However, examination of the folding and assembly of subunits of form I Rubisco from certain cyanobacteria indicated that there was more to the story of Rubisco assembly. In the cyanobacterium Anabaena and in other species, the rbcX gene is situated between rbcL and rbcS (Larimer and Soper, 1993Larimer F.W. Soper T.S. Gene. 1993; 126: 85-92Crossref PubMed Scopus (30) Google Scholar), and all three genes are cotranscribed. Most importantly, RbcX is important for maximal production of catalytically competent recombinant cyanobacterial Rubisco (Li and Tabita, 1997Li L.-A. Tabita F.R. J. Bacteriol. 1997; 179: 3793-3796Crossref PubMed Google Scholar), although its function has been unclear. Curiously, rbcX is either required or nonessential for Rubisco activity and solubility depending on the strain of unicellular cyanobacterium (Onizuka et al., 2004Onizuka T. Endo S. Akiyama H. Kanai S. Hirano M. Yokota A. Tanaka S. Miyasaka H. Plant Cell Physiol. 2004; 45: 1390-1395Crossref PubMed Scopus (36) Google Scholar, Emlyn-Jones et al., 2006Emlyn-Jones D. Woodger F.J. Price C.D. Whitney S.M. Plant Cell Physiol. 2006; 47: 1630-1640Crossref PubMed Scopus (47) Google Scholar). In an elegant new study, Saschenbrecker et al., 2007Saschenbrecker S. Bracher A. Rao K.V. Rao B.V. Hartl F.U. Hayer-Hartl M. Cell. 2007; (this issue)PubMed Google Scholar present biochemical and structural evidence that RbcX acts as a specific Rubisco assembly chaperone to facilitate formation of an oligomeric RbcL8 ((RbcL2)4) core complex. Newly formed RbcL8 is then capable of spontaneously combining with S subunits to form the catalytically competent hexadecameric holoenzyme complex. RbcX performs this feat after GroEL and GroES, and perhaps other chaperones, have assisted productive folding of newly translated Rubisco L polypeptides. Soon after folded RbcL emerges, RbcX maximizes the correct assembly of RbcL octamers; otherwise, RbcL has a tendency to misassemble or produce unproductive aggregates. To gain further insight into how RbcX functions, Saschenbrecker and colleagues solved X-ray structures of two separate RbcX proteins. These efforts yielded similar structures, suggesting that all RbcX proteins are functionally alike. The asymmetric unit appears to be composed of three dimers. Two highly conserved regions were identified, one at the central groove of the dimer, which contains several hydrophobic residues. The other conserved region is a polar surface region, located around the corners of the molecule. Site-directed mutagenesis of various residues in the two regions indicates that the central groove region is essential and facilitates the formation of soluble RbcL subunits, whereas the polar surface region ensures that the RbcL subunits are properly arranged. The structural studies also indicate that the central groove of RbcX could serve as an interface for binding a long peptide (presumably derived from newly translated RbcL). The authors provide evidence that a conserved peptide (EIKFEFD), derived from the C terminus of RbcL, specifically interacts with the central groove of RbcX in a dynamic manner. The last stage of the assembly process involves the displacement of RbcX from the RbcL-RbcX complex by RbcS, resulting in the formation of the functional hexadecameric holoenzyme. Clearly, these studies represent a seminal advance toward understanding how a complex oligomeric protein assembles in the cell. Spurred by this work, others will undoubtedly seek to determine whether similar systems and other chaperones for protein assembly might have functions analogous to RbcX. This study provokes many questions and also reveals additional curiosities. First of all, the authors suggest that RbcX might play a general role in the assembly of form I Rubiscos, which have been subdivided into four distinct classes (A through D). RbcX may play this role in cyanobacteria, green algae, and plants, all of which possess RbcX homologs and form IB Rubisco (Tabita, 1999Tabita F.R. Photosynth. Res. 1999; 60: 1-28Crossref Scopus (271) Google Scholar). However, many organisms, such as proteobacteria and nongreen algae that contain form I Rubisco (forms IC and ID), do not possess any discernible RbcX homologs. In addition, the genes encoding the IC form of Rubisco from purple nonsulfur photosynthetic bacteria and various chemolithoautotrophic bacteria are easily expressed as fully functional hexadecameric proteins in Escherichia coli without the need for any other protein. Interestingly, comparisons of the C-terminal regions of several form I Rubiscos indicate that the form IC proteobacterial proteins, as well as form ID enzymes from nongreen algae, do not contain the absolute consensus C-terminal RbcX recognition motif (EIKFEFD) found in form IB enzymes (Table 1). For the IC and ID forms of Rubisco, there are two conserved residues and a couple of conservative substitutions in this region. Is this enough to confer specificity for any type of RbcX protein? Form IA Rubisco, found in some marine cyanobacteria and other classes of chemolithotrophic proteobacteria, does contain this motif, yet, to date, no identifiable RbcX homologs have been identified in these organisms. Does this mean that even though these organisms contain the RbcX recognition motif at the Rubisco L C terminus, they use another protein to facilitate assembly? As for organisms that contain the IC form of Rubisco but do not possess the consensus C-terminal motif of IB Rubisco or an RbcX protein, is there a completely independent mechanism to facilitate assembly of the enzyme both in the native organism and in E. coli? Is it possible that these enzymes do not require any type of assembly chaperone? Finally, eukaryotic nongreen algae, which contain form ID Rubisco and no identifiable RbcX homolog, are similar to bacteria in that both the rbcL and rbcS genes are cotranscribed behind a single promoter, but in the chloroplast of these organisms. As with other eukaryotic Rubisco genes, active fully assembled recombinant form ID Rubisco has not been successfully produced in E. coli, suggesting that additional factors and/or specific processing/posttranslational events may be required. Although, as the authors suggest, it might be necessary to take RbcX into account for future attempts to improve the catalytic properties of Rubisco in crop plants, the new work raises other intriguing questions concerning assembly of Rubisco, the most abundant enzyme known.Table 1The RbcX Recognition Motif in the Large Subunit of RubiscoRubisco TypeOrganismC-Terminal SequenceIBSynechococcus PCC7002EIKFEFDIBSynechococcus PCC6301EIKFEFEIBAnabaena PCC7210EIKFEFEIBChlamydomonas reinhardtiiEIKFEFDIBSpinacia oleraceaEIKFEFPIAThiobacillus denitrificansEIKFEFDIASynechococcus WH8102EIKFEFDIAProchlorococcus MIT9312EIKFEFDICRalstonia eutropha H16DISFNYTICRhodobacter sphaeroides 241NITFNYTICXanthobacter flavusEVTFNYAIDCylindrotheca N1DISFNYTIDPorphyridium aerugineumDISFNYTIDCryptomonas ϕDITFNYAForm I Rubisco can be subdivided into four distinct classes (A, B, C, and D) based on sequence homology (Tabita, 1999Tabita F.R. Photosynth. Res. 1999; 60: 1-28Crossref Scopus (271) Google Scholar). The EIKFEFD RbcX interaction motif is found at the C terminus of RbcL/CbbL in organisms containing form IB and form IA Rubisco. However, to date, only organisms containing form IB Rubisco are known to have the RbcX protein. With only two residues in common, along with a couple of conservative substitutions, form IC and form ID Rubisco possess sequence differences at their C termini. Organisms containing these classes of form I Rubisco do not contain any discernible RbcX homolog. These sequence differences and the absence of RbcX in some organisms suggest that the mechanisms to assemble form I Rubisco hexadecamers may differ depending on the organism. Open table in a new tab Form I Rubisco can be subdivided into four distinct classes (A, B, C, and D) based on sequence homology (Tabita, 1999Tabita F.R. Photosynth. Res. 1999; 60: 1-28Crossref Scopus (271) Google Scholar). The EIKFEFD RbcX interaction motif is found at the C terminus of RbcL/CbbL in organisms containing form IB and form IA Rubisco. However, to date, only organisms containing form IB Rubisco are known to have the RbcX protein. With only two residues in common, along with a couple of conservative substitutions, form IC and form ID Rubisco possess sequence differences at their C termini. Organisms containing these classes of form I Rubisco do not contain any discernible RbcX homolog. These sequence differences and the absence of RbcX in some organisms suggest that the mechanisms to assemble form I Rubisco hexadecamers may differ depending on the organism. Structure and Function of RbcX, an Assembly Chaperone for Hexadecameric RubiscoSaschenbrecker et al.CellJune 15, 2007In BriefAfter folding, many proteins must assemble into oligomeric complexes to become biologically active. Here we describe the role of RbcX as an assembly chaperone of ribulose-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme responsible for the fixation of atmospheric carbon dioxide. In cyanobacteria and plants, Rubisco is an ∼520 kDa complex composed of eight large subunits (RbcL) and eight small subunits (RbcS). We found that cyanobacterial RbcX functions downstream of chaperonin-mediated RbcL folding in promoting the formation of RbcL8 core complexes. Full-Text PDF Open Access
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