Structural insights into the functions of Raf1 and Bsd2 in hexadecameric Rubisco assembly
Ran Wang, Hui Song, Wenjuan Zhang, Ning Wang, Shijia Zhang, Ruiqi Shao, Cuimin Liu
Abstract
Hexadecameric form I Rubisco, which consisting consists of eight large (RbcL) and eight small (RbcS) subunits, is the most abundant enzyme on earth. Extensive efforts to engineer an improved Rubisco to speed up its catalytic efficiency and ultimately increase agricultural productivity. However, difficulties with correct folding and assembly in foreign hosts or in vitro have hampered the genetic manipulation of hexadecameric Rubisco. In this study, we reconstituted Synechococcus sp. PCC6301 Rubisco in vitro using the chaperonin system and assembly factors from cyanobacteria and Arabidopsis thaliana ( At ). Rubisco holoenzyme was produced in the presence of cyanobacterial Rubisco accumulation factor 1 (Raf1) alone or both At Raf1 and bundle-sheath defective-2 ( At Bsd2) from Arabidopsis . RbcL released from GroEL is assembly capable in the presence of ATP, and At Bsd2 functions downstream of At Raf1. Cryo-EM structures of RbcL 8 – At Raf1 8 , RbcL 8 – At Raf1 4 – At Bsd2 8 , and RbcL 8 revealed that the interactions between RbcL and At Raf1 are looser than those between prokaryotic RbcL and Raf1, with At Raf1 tilting 7° farther away from RbcL. At Bsd2 stabilizes the flexible regions of RbcL, including the N and C termini, the 60s loop, and loop 6. Using these data, combined with previous findings, we propose the possible biogenesis pathways of prokaryotic and eukaryotic Rubisco.