Role of Small Subunit in Mediating Assembly of Red - type Form 1 Rubisco *

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into greenand red-type. Some redtype enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the greentype RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone independent manner. We find that the C-terminal -hairpin extension of red-type RbcSwhich is absent in green-type RbcSis critical for efficient assembly. The -hairpins of four RbcS subunits form a 8-stranded -barrel that protrudes into the central solvent channel of the RbcL core complex. The two -barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal -hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing redtype Rubisco. The enzyme Rubisco catalyzes the carboxylation of the five-carbon sugar ribulose 1,5-bisphosphate (RuBP) in the dark reactions of photosynthesis, thereby mediating the conversion of atmospheric CO2 into usable sugars. In its most abundant form (form I), Rubisco is a hexadecameric complex composed of 8 large (RbcL, ~50 kDa) and 8 small subunits (RbcS, ~1215 kDa) (1,2). The RbcL subunits form a cylindrical tetramer of anti-parallel dimers that is capped by four RbcS subunits at the top and four JBC C o n f i d e n t i a l Red-type Rubisco assembly