Thylakoid luminal θ-carbonic anhydrase critical for growth and photosynthesis in the marine diatom Phaeodactylum tricornutum

Abstract

The algal pyrenoid is a large plastid body, where the majority of the CO 2 -fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) resides, and it is proposed to be the hub of the algal CO 2 -concentrating mechanism (CCM) and CO 2 fixation. The thylakoid membrane is often in close proximity to or penetrates the pyrenoid itself, implying there is a functional cooperation between the pyrenoid and thylakoid. Here, GFP tagging and immunolocalization analyses revealed that a previously unidentified protein, Pt43233, is targeted to the lumen of the pyrenoid-penetrating thylakoid in the marine diatom Phaeodactylum tricornutum. The recombinant Pt43233 produced in Escherichia coli cells had both carbonic anhydrase (CA) and esterase activities. Furthermore, a Pt43233:GFP-fusion protein immunoprecipitated from P. tricornutum cells displayed a greater specific CA activity than detected for the purified recombinant protein. In an RNAi-generated Pt43233 knockdown mutant grown in atmospheric CO 2 levels, photosynthetic dissolved inorganic carbon (DIC) affinity was decreased and growth was constantly retarded; in contrast, overexpression of Pt43233:GFP yielded a slightly greater photosynthetic DIC affinity. The discovery of a θ-type CA localized to the thylakoid lumen, with an essential role in photosynthetic efficiency and growth, strongly suggests the existence of a common role for the thylakoid-luminal CA with respect to the function of diverse algal pyrenoids. marine diatom | CGHR domain | luminal carbonic anhydrase | CO 2 -concentrating mechanism | pyrenoid M arine diatoms are major primary producers, which are responsible for up to 20% of annual global carbon fixation (1, 2). To overcome the difficulties of CO 2 limitation in alkaline and high-salinity seawater, diatoms use a CO 2 -concentrating mechanism (CCM) for the intracellular accumulation of dissolved inorganic carbon (DIC) (3). It is known that the marine pennate diatom, Phaeodactylum tricornutum, uses solute carrier 4 (SLC4) family transporters to take up HCO 3 − actively from the surrounding seawater (4). Based upon physiological measurements of cellular DIC flux, it has been hypothesized that accumulated HCO 3 − is further concentrated in the chloroplast and that an ample flux of CO 2 to ribulose-1,5-bisphosphate carboxylase/ oxygenase (RubisCO) is facilitated by the pyrenoidal β-carbonic anhydrases (CAs), PtCA1 and PtCA2 (5, 6). In this process, α-type CAs present in the matrices of the four-layered chloroplast membranes are thought to prevent leakage of CO 2 from the chloroplast in P. tricornutum (7,8).Algal CCMs are distinct from their carboxysomal counterparts in cyanobacteria, and were most likely acquired by an extensive convergent evolution process (9). It is postulated that the algal CCM is composed of active DIC transport systems at the plasma membrane and the chloroplast envelope, as well as a highly localized CO 2 formation system within close proximity to RubisCO. The possibility remains that the latter process occurs within the py...

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

“…This indicates that the difference in their activity is likely a result of substrate binding or accessibility of the active site, considering the competitive nature of the inhibitor. Recently, Matsuda et al (27) also demonstrated that PtLCIB3 (designated in their work as Pt43233) has CA activity and is localized to the thylakoid membrane spanning the pyrenoid. To understand the molecular basis of the observed biochemical results, we set out to structurally characterize the various LCIB proteins.…”

“…Thus, the bicarbonate-dependent activation mechanism is unlikely to be present in these proteins. Regarding the function of the non-C. reinhardtii proteins analyzed here, Kikutani et al (27) recently published a key study regarding the localization and function of PtLCIB3. They reported that in the diatom, this gene product localizes to the lumen of pyrenoid-penetrating thylakoid and is essential for the CCM function of the algae.…”

Phenols and Polyphenols as Carbonic Anhydrase Inhibitors

“…To date seven not-genetically related CA families are reported, indicated with the Greek letters α-, β-, γ-, δ-, ζ-η-and θ- [1][2][3][4][5][6][7][8][9]. All the CA families have been thoroughly characterized by means of kinetic and structural features, with the exception of the δ-, η-and θ-CAs, for which only kinetic data are available so far [8][9][10][11]. The α-CAs are mainly distributed among the vertebrates, protozoa, algae, cytoplasm of green plants and in many Bacteria species.…”

Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations

“…Based on their structural features, they are grouped into seven different classes, namely a-, b-, c-, d-, f-, g-and h-CAs. a-CAs are predominantly expressed in vertebrates, bacteria, algae and cytoplasm of green plants, b-CAs in bacteria, algae and chloroplasts, c-CAs in archaea and some bacteria, d-and f-CAs in some marine diatoms, g-CAs only in the protozoan parasite Plasmodium spp., whereas the recently discovered h-class has been so far found only into the marine diatom Phaeodactylum tricornutum [1][2][3][4][5][6][7][8] . Humans encode 12 catalytically active a-CA isozymes, which differ in molecular features, oligomeric arrangement, kinetic properties and cellular localisation, with isoforms I, II, III, VII and XIII localised in the cytosol, CA IV, IX, XII and XIV associated with the cell membrane, CA VA and VB confined in mitochondria, and CA VI secreted in saliva and milk 1 .…”