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 Kombinatori Tertukar 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

 

Received: 14 March 2018/Accepted: 4 June 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

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*Corresponding author; email: bhlim@utar.edu.my

 

 

 

 

 

 

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