Rubisco functional integrity requires a dedicated three-step maturation pathway.

Abstract

Proteins generally begin with methionine, which is often removed to expose the second residue. RuBP carboxylase/oxygenase (Rubisco), the most abundant enzyme on Earth and a major carbon and nitrogen reservoir, escapes this paradigm. Its catalytic subunit, RbcL, undergoes unusual amino terminal maturation, including atypical initiator methionine-serine cleavage and proline acetylation, a modification not seen in other biological systems. We define a specialized pathway of two sequential aminopeptidases and an amino terminal acetyltransferase, dedicated to RbcL, that executes processing. This maturation is coupled to chaperone-assisted holoenzyme assembly, enhances activation by Rubisco activase, and shields the complex from degradation during senescence. Disruption of this pathway reduces Rubisco accumulation and catalytic competence, revealing an evolutionarily tailored mechanism integrating protein maturation, assembly, and stability of a central metabolic enzyme.