Structure and Identification of a Pterin Dehydratase-like Protein as a Ribulose-bisphosphate Carboxylase/Oxygenase (RuBisCO) Assembly Factor in the α-Carboxysome

Background: A gene encoding a distant homolog of pterin-4-carbinolamine dehydratase (PCD) is present in all -type carboxysome operons. Results: This conserved PCD-like protein promotes the assembly of native RuBisCO when overexpressed together with GroELS. Conclusion: The protein, named here acRAF for -carboxysome RuBisCO assembly factor, is a newly identified molecular chaperone. Significance: The activity of this chaperone toward RuBisCO has potential agricultural application. Carboxysomes are proteinaceous bacterial microcompartments that increase the efficiency of the rate-limiting step in carbon fixation by sequestering reaction substrates. Typically, -carboxysomes are genetically encoded as a single operon expressing the structural proteins and the encapsulated enzymes of the microcompartment. In addition, depending on phylogeny, as many as 13 other genes are found to co-occur near or within -carboxysome operons. One of these genes codes for a protein with distant homology to pterin-4-carbinolamine dehydratase (PCD) enzymes. It is present in all -carboxysome containing bacteria and has homologs in algae and higher plants. Canonical PCDs play an important role in amino acid hydroxylation, a reaction not associated with carbon fixation. We determined the crystal structure of an -carboxysome PCD-like protein from the chemoautotrophic bacterium Thiomonas intermedia K12, at 1.3- resolution. The protein retains a three-dimensional fold similar to canonical PCDs, although the prominent active site cleft present in PCD enzymes is disrupted in the -carboxysome PCD-like protein. Using a cell-based complementation assay, we tested the PCD-like proteins from T. intermedia and two additional bacteria, and found no evidence for PCD enzymatic activity. However, we discovered that heterologous co-expression of the PCD-like protein from Halothiobacillus neapolitanus with RuBisCO and GroELS in Escherichia coli increased the amount of soluble, assembled RuBisCO recovered from cell lysates compared with co-expression of RuBisCO with GroELS alone. We conclude that this conserved PCD-like protein, renamed here -carboxysome RuBisCO assembly factor (or acRAF), is a novel RuBisCO chaperone integral to -carboxysome function.