Sains Malaysiana 47(10)(2018): 2269–2289

http://dx.doi.org/10.17576/jsm-2018-4710-04

 

Dissection of Synechococcus Rubisco Large Subunit Sections Involved in Holoenzyme Formation in Escherichia coli by Combinatorial Section Swapping and Sequence Analyses

(Pembahagian Synechococcus Rubisco Seksyen Subunit Besar Terlibat dalam Pembentukan holoenzim dalam Escherichia coli oleh Seksyen KombinatoriTertukar dan Jujukan Analisis)

 

YEE HUNG YEAP1, TENG WEI KOAY1, HANN LING WONG2 & BOON HOE LIM1*

 

1Department of Chemical Science, Universiti Tunku Abdul Rahman, 31900 Kampar, Perak Darul Ridzuan, Malaysia

 

2Department of Biological Science, Universiti Tunku Abdul Rahman, 31900 Kampar, Perak Darul Ridzuan, Malaysia

 

Diserahkan: 14 Mac 2018/Diterima: 4 Jun 2018

 

ABSTRACT

Engineering the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to improve photosynthesis has long been sought. Rubisco large subunits (RbcL) are highly-conserved but because of certain undefined sequence differences, plant Rubisco research cannot fully utilise the robust heterologous Escherichia coli expression system and its GroEL folding machinery. Previously, a series of chimeric cyanobacteria Synechococcus elongatus Rubisco, incorporated with sequences from the green alga Chlamydomonas reinhardtii, were expressed in E. coli; differences in RbcL sections essential for holoenzyme formation were pinpointed. In this study, the remaining sections, presumably not crucial for holoenzyme formation and also the small subunit (RbcS), are substituted to further ascertain the possible destabilising effects of multiple section mutations. To that end, combinations of Synechococcus RbcL Sections 1 (residues 1-47), 2 (residues 48-97), 5 (residues 198-247) and 10 (residues 448-472), and RbcS, were swapped with collinear Chlamydomonas sections and expressed in E. coli. Interestingly, only the chimera with Sections 1 and 2 together produces holoenzyme and an interaction network of complementing amino acid changes is delineated by crystal structure analysis. Furthermore, sequence-based analysis also highlighted possible GroEL binding site differences between the two RbcLs.

 

Keywords: Chaperone; Chlamydomonas reinhardtii; protein assembly; ribulose bisphosphate carboxylase/oxygenase (Rubisco); Synechococcus elongatus PCC6301

 

ABSTRAK

Kajian untuk mengubah suai ribulosa-1,5-bisfosfat karboksilase/oksigenase (Rubisco) bagi memperbaiki proses fotosintesis adalah usaha yang telah lama dijalankan. Subunit- besar Rubisco amat konservatif tetapi disebabkan perbezaan jujukan asid amino yang tertentu, Rubisco tumbuh-tumbuhan tidak dapat dikaji dengan menggunakan sistem pengekspresan Escherichia coli yang serba-boleh serta mekanisme penglipatan GroEL-nya. Sebelum ini, satu siri Rubisco kimerik yang menggabungkan jujukan daripada cyanobacteria Synechococcus elongatus dengan alga hijau Chlamydomonas reinhardtii telah diekspreskan ke dalam E. coli; dalam uji kaji tersebut, perbezaan yang merangkumi seksyen RbcL yang mustahak dalam pembentukan holoenzim telah ditentukan. Dalam uji kaji ini, seksyen lain yang mungkin tidak penting untuk pembentukan holoenzim, bersama-sama subunit kecil (RbcS) telah digantikan untuk menentukan kemungkinan kesan ketidakstabilan akibat mutasi seksyen berbilang. Untuk itu, kombinasi Synechococcus RbcL Seksyen 1 (residu 1-47), 2 (residu 48-97), 5 (residu 198-247) dengan 10 (residu 448-472) dan RbcS, telah digantikan dengan seksyen Chlamydomonas yang kolinear dan diekspreskan dalam E. coli. Kesimpulannya, hanya kimera yang ditukarkan kedua-dua Seksyen 1 dan 2 dapat membentuk holoenzim dan rangkaian interaksi yang meliputi perubahan asid amino yang saling melengkapkan berdasarkan analisis struktur kristal telah dikemukakan. Selain itu, analisis berasaskan jujukan asid amino juga menunjukkan bahawa perbezaan tapak ikatan GroEL yang mungkin bagi RbcL.

 

Kata kunci: Chaperone; Chlamydomonas reinhardtii; himpunan protein; ribulosa-1,5-bisfosfat karboksilase/oksigenase (Rubisco); Synechococcus elongatus PCC6301

RUJUKAN

Aigner, H., Wilson, R.H., Bracher, A. Calisse, L., Bhat, J.Y., Hartl, F.U. & Hayer-Hartl, M. 2017. Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2. Science 358: 1272-1278.

Andersson, I. & Backlund, A. 2008. Structure and function of Rubisco. Plant Physiology and Biochemistry 46: 275-291.

Bainbridge, G., Madgwick, P.J., Parmar, S., Mitchell, R., Paul, M., Pitts, J., Keys, A.J. & Parry, M.A.J. 1995. Engineering Rubisco to change its catalytic properties. Journal of Experimental Botany 46: 1269-1276.

Bloom, J.D., Silberg, J.J., Wilke, C.O., Drummond, D.A., Adami, C. & Arnold, F.H. 2005. Thermodynamic prediction of protein neutrality. Proceedings of the National Academy of Science of the United States of America 102: 606-611.

Burger, R., Willensdorfer, M. & Nowak, M.A. 2006. Why are phenotypic mutation rates much higher than genotypic mutation rates? Genetics 172: 197-206.

Campbell, W.J. & Ogren, W.L. 1992. Light activation of Rubisco by Rubisco activase and thylakoid membranes. Plant Cell Physiology 33: 751-756.

Chaudhuri, T.K. & Gupta, P. 2005. Factors governing the substrate recognition by GroEL chaperone: A sequence correlation approach. Cell Stress Chaperones 10: 24-36.

Chen, Z. & Spreitzer, R.J. 1992. How various factors influence the CO2/O2 specificity of ribulose-1, 5-bisphosphate carboxylase/ oxygenase. Photosynthesis Research 31: 157-164.

Cloney, L.P., Bekkaoui, D.R. & Hemmingsen, S.M. 1993. Co-expression of plastid chaperonin genes and a synthetic plant Rubisco operon in Escherichia coli. Plant Molecular Biology 23: 1285-1290.

Cohen, I., Sapir, Y. & Shapira, M. 2006. A conserved mechanism controls translation of Rubisco large subunit in different photosynthetic organisms. Plant Physiology 141: 1089-1097.

Cordes, M.H. & Sauer, R.T. 1999. Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions. Protein Science 8: 318-325.

Curmi, P.M.G., Cascio, D., Sweet, R.M., Eisenberg, D. & Schreuder, H. 1992. Crystal structure of the unactivated form of ribulose- 1,5-bisphosphate carboxylase/ oxygenase from tobacco refined at 20-Å resolution. Journal of Biological Chemistry 267: 16980-16989.

Ellis, R.J. 1979. The most abundant protein in the world. Trends in Biochemical Science 4: 241-244.

Esquivel, M.G., Genkov, T., Nogueira, A.S., Salvucci, M.E. & Spreitzer, R.J. 2013. Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O2 specificity but not activation by Rubisco activase. Photosynthesis Research 118: 209-218.

Feller, U., Anders, I. & Mae, T. 2008. Rubiscolytics: Fate of Rubisco after its enzymatic function in a cell is terminated. Journal of Experimental Botany 59: 1615-1624.

Genkov, T., Du, Y.C. & Spreitzer, R.J. 2006. Small subunit cysteine-65 substitutions can suppress or induce alterations in the large-subunit catalytic efficiency and holoenzyme thermal stability of ribulose-1,5-bisphosphate carboxylase/oxygenase. Archives of Biochemistry and Biophysics 451: 167-174.

Genkov, T. & Spreitzer, R.J. 2009. Highly conserved small subunit residues influence Rubisco large subunit catalysis. Journal of Biological Chemistry 284: 30105-30112.

Genkov, T., Meyer, M., Griffiths, H. & Spreitzer, R.J. 2010. Functional hybrid Rubisco enzymes with plant small subunits and algal large subunits: Engineered rbcS cDNA for expression in Chlamydomonas. Journal of Biological Chemistry 285: 19833-19841.

Goloubinoff, P., Gatenby, A.A. & Lorimer, G.H. 1989. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature 337: 44-47.

Gutteridge, S., Rhoades, D.F. & Herrmann, C. 1993. Site-specific mutations in a loop region of the C-terminal domain of the large subunit of ribulose bisphosphate carboxylase/oxygenase that influence substrate partitioning. Journal of Biological Chemistry 268: 7818-7824.

Jordan, D.B. & Ogren, W.L. 1981. Species variation in the specificity of ribulose biphosphate carboxylase/oxygenase. Nature 291: 513-515.

Kannappan, B. & Gready, J.E. 2008. Redefinition of Rubisco carboxylase reaction reveals origin of water for hydration and new roles for active-site residues. Journal of the American Chemical Society 130: 15063-15080.

Knight, S., Andersson, I. & Brändén, C.I. 1990. Crystallographic analysis of ribulose 1,5-bisphosphate carboxylase from spinach at 2·4 Å resolution. Journal of Molecular Biology 215: 113-160.

Koay, T.W., Wong, H.L. & Lim, B.H. 2016. Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in Escherichia coli. Genes & Genetic Systems 91: 139-150.

Kumar, V., Punetha, A., Sundar, D. & Chaudhuri, T.K. 2012. In silico engineering of aggregation-prone recombinant proteins for substrate recognition by the chaperonin GroEL. BMC Genomics 13: S22.

Kyte, J. & Doolittle, R.F. 1982. A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157: 105-132.

Laing, W.A., Ogren, W.L. & Hageman, R.H. 1974. Regulation of soybean net photosynthetic CO2 fixation by the interaction of CO2, O2, and ribulose 1,5-diphosphate carboxylase. Plant Physiology 54: 678-685.

Lin, Z. & Rye, H.S. 2006. GroEL-mediated protein folding: Making the impossible, possible. Critical Reviews in Biochemistry and Molecular Biology 41: 211-239.

Long, S.P., Zhu, X-G., Naidu, S.L. & Ort, D.R. 2006. Can improvement in photosynthesis increase crop yields? Plant, Cell and Environment 29: 315-330.

Marin-Navarro, J. & Moreno, J. 2006. Cysteines 449 and 459 modulate the reduction-oxidation conformational changes of ribulose 1.5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress. Plant, Cell and Environment 29: 898-908.

Meyer, M.T., Genkov, T., Skepper, J.N., Jouhet, J., Mitchell, M.C., Spreitzer, R.J. & Griffiths, H. 2012. Rubisco small-subunit α-helices control pyrenoid formation in Chlamydomonas. Proceedings of the National Academy of Science of the United States of America 109: 19474-19479.

Mueller-Cajar, O. & Whitney, S.M. 2008. Evolving improved Synechococcus Rubisco functional expression in Escherichia coli. Biochemical Journal 414: 205-214.

Ogren, W.L. 1984. Photorespiration: Pathways, regulation, and modification. Annual Review of Plant Physiology and Plant Molecular Biology 35: 415-442.

Ott, C.M., Smith, B.D., Portis, A.R. & Spreitzer, R.J. 2000. Activase region on chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. Journal of Biological Chemistry 275: 26241-26244.

Pace, C.N. 2001. Polar group burial contributes more to protein stability than nonpolar group burial. Biochemistry 40: 310- 313.

Pakula, A.A. & Sauer, R.T. 1990. Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface. Nature 344: 363-364.

Parikh, M.R., Greene, D.A., Woods, K.K. & Matsumura, I. 2006. Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E. coli. Protein Engineering, Design and Selection 19: 113-119.

Parry, M.A.J., Madgwick, P.J., Carvalho, J.F.C. & Andralojc, P.J. 2007. Prospects for increasing photosynthesis by overcoming the limitations of Rubisco. Journal of Agricultural Science 145: 31-43.

Peterhansel, C., Niessen, M. & Kebeish, R.M. 2008. Metabolic engineering towards the enhancement of photosynthesis. Photochemistry and Photobiology 84: 1317-1323.

Rasineni, G.K., Loh, P.C. & Lim, B.H. 2017. Characterization of Chlamydomonas ribulose-1,5-bisphosphate carboxylase/ oxygenase variants mutated at residues that are post-translationally modified. Biochimica et Biophysica Acta 1861(2): 79-85.

Saschenbrecker, S., Bracher, A., Rao, K.V., Rao, B.V., Hartl, F.U. & Hayer-Hartl, M. 2007. Structure and function of RbcX, an assembly chaperone for hexadecameric Rubisco. Cell 129: 1189-1200.

Smith, S.A. & Tabita, F.R. 2003. Positive and negative selection of mutant forms of prokaryotic (cyanobacterial) ribulose-1,5- bisphosphate carboxylase/oxygenase. Journal of Molecular Biology 331: 557-569.

Spreitzer, R.J., Thow, G. & Zhu, G. 1995. Pseudoreversion substitution at large-subunit residue 54 influences the CO2/O2 specificity of chloroplast ribulose bisphosphate carboxylase/ oxygenase. Plant Physiology 109: 681-685.

Stan, G., Brooks, B.R., Lorimer, G.H. & Thirumalai, D. 2006. Residues in substrate proteins that interact with GroEL in the capture process are buried in the native state. Proceedings of the National Academy of Science of the United States of America 103: 4433-4438.

Stan, G., Brooks, B.R., Lorimer, G.H. & Thirumalai, D. 2004. Identifying natural substrates for chaperonins using a sequence-based approach. Protein Science 14: 193-201.

Tabita, F.R., Hanson, T.E., Satagopan, S., Witte, B.H. & Kreel, N.E. 2008. Phylogenetic and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms. Philosophical Transactions of the Royal Society B 363: 2629-2640.

Tabita, F.R., Hanson, T.E., Li, H., Satagopan, S., Singh, J. & Chan, S. 2007a. Function, structure, and evolution of the RubisCO-like proteins and their RubisCO homologs. Microbiology and Molecular Biology Reviews 71(4): 576-599.

Tabita, F.R., Satagopan, S., Hanson, T.E., Kreel, N.E. & Scott, S.S. 2007b. Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships. Journal of Experimental Botany 59: 1515-1524.

Takano, K., Yamagata, Y. & Yutani, K. 2001. Contribution of polar groups in the interior of a protein to the conformational stability. Biochemistry 40: 4853-4858.

Taylor, T.C., Backlund, A., Bjorhall, K., Spreitzer, R.J. & Andersson, I. 2001. First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. Journal of Biological Chemistry 276: 48159-48164.

Tcherkez, G.G.B., Farquhar, G.D. & Andrews, T.J. 2006. Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proceedings of the National Academy of Science of the United States of America 103: 7246-7251.

Whitney, S.M., Baldet, P., Hudson, G.S. & Andrews, T.J. 2001. Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts. Plant Journal 26: 535-547.

Whitney, S.M., Houtz, R.L. & Alonso, H. 2011. Advancing our understanding and capacity to engineer nature’s CO2- sequestering enzyme, Rubisco. Plant Physiology 155: 27-35.

Wilson, R.H., Martin-Avila, E., Conlan, C. & Whitney, S.M. 2017. An improved Escherichia coli screen for Rubisco identifies a protein-protein interface that can enhance CO2- fixation kinetics. Journal of Biological Chemistry 293: 18-27.

Wostrikoff, K. & Stern, D. 2007. Rubisco large-subunit translation is autoregulated in response to its assembly state in tobacco chloroplasts. Proceedings of the National Academy of Science of the United States of America 104: 6466-6471.

Zhu, X.G., Portis, A.R. & Long, S.P. 2004. Would transformation of C3 crop plants with foreign Rubisco increase productivity? A computational analysis extrapolating from kinetic properties to canopy photosynthesis. Plant, Cell and Environment 27: 155-165.

 

 

*Pengarang untuk surat-menyurat; email: bhlim@utar.edu.my

 

 

 

 

 

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