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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;
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