Structural insights into the LCIB protein family reveals a new group of β-carbonic anhydrases

Abstract

Aquatic microalgae have evolved diverse CO 2 -concentrating mechanisms (CCMs) to saturate the carboxylase with its substrate, to compensate for the slow kinetics and competing oxygenation reaction of the key photosynthetic CO 2 -fixing enzyme rubisco. The limiting CO 2 -inducible B protein (LCIB) is known to be essential for CCM function in Chlamydomonas reinhardtii. To assign a function to this previously uncharacterized protein family, we purified and characterized a phylogenetically diverse set of LCIB homologs. Three of the six homologs are functional carbonic anhydrases (CAs). We determined the crystal structures of LCIB and limiting CO 2 -inducible C protein (LCIC) from C. reinhardtii and a CA-functional homolog from Phaeodactylum tricornutum, all of which harbor motifs bearing close resemblance to the active site of canonical β-CAs. Our results identify the LCIB family as a previously unidentified group of β-CAs, and provide a biochemical foundation for their function in the microalgal CCMs.LCIB | limiting-CO 2 inducible protein | CO 2 -concentrating mechanism | photosynthesis | carbonic anhydrases

A Spatial Interactome Reveals the Protein Organization of the Algal CO2-Concentrating Mechanism

“…The pyrenoid matrix contains the Rubisco holoenzyme (RBCS/RbcL); its chaperone Rubisco activase (RCA1); essential pyrenoid component 1 (EPYC1), a Rubisco linker protein important for Rubisco packaging in the pyrenoid (Mackinder et al, 2016); and a protein of unknown function (Cre06.g259100; Kobayashi et al, 2016). Under very low CO 2 conditions, the LCIB/LCIC complex, whose role is still uncertain (Jin et al, 2016), is known to form puncta around the pyrenoid periphery (Yamano et al, 2010). Recently, a Ca 2+ -binding protein, CAS, has been shown to specifically localize to the pyrenoid tubules at low CO 2 (Wang et al, 2016).…”

“…A role in the conversion of CO 2 to HCO 3 − is likely, as several homologs of LCIB were recently shown to be functional β-carbonic anhydrases. However, recombinant LCIB/C had no carbonic anhydrase function (Jin et al, 2016), suggesting that the complex may be tightly regulated or may require additional factors for proper function.…”

CO2‐fixing liquid droplets: Towards a dissection of the microalgal pyrenoid

“…CO 2 then diffuses across the thylakoid membrane to the proximate Rubisco active sites and is fixed into 3‐phosphoglycerate. LCIB/LCIC, which structurally resembles a carbonic anhydrase, localizes peripheral to the pyrenoid matrix and is hypothesized to function in recapture (hydration) of CO 2 leaking from the pyrenoid . Another prominent feature of the Chlamydomonas pyrenoid is a starch sheath that physically surrounds the compartment and is formed in response to low CO 2 concentrations .…”

“…In a similar manner, the in vitro assay allows us to test whether completely unrelated control proteins or proteins known to be absent from the pyrenoid matrix (for instance, other Calvin cycle enzymes or the predicted carbonic anhydrase LCIB/LCIC) are actively excluded from the droplets or whether their in vivo localization will be determined by other mechanisms. Mackinder et al reported that proteins exceeding 78 kDa appear to be absent from the pyrenoid matrix, which could be caused by the surface tension of the liquid phase .…”

The Chlamydomonas CO2‐concentrating mechanism and its potential for engineering photosynthesis in plants

“…The other two Ci flux-associated proteins are CAH3, a thylakoid lumen-localized carbonic anhydrase; and LCIB found in the chloroplast stroma. Although the precise function of LCIB is unclear, LCIB orthologs in other algae function as carbonic anhydrases that are perhaps involved in CO 2 recapture by directional hydration of CO 2 to HCO 3 À (Jin et al, 2016;Kikutani et al, 2016). limiting CO 2 (Turkina et al, 2006;Blanco-Rivero et al, 2012;.…”