RESUMEN
Aquatic microalgae have evolved diverse CO2-concentrating mechanisms (CCMs) to saturate the carboxylase with its substrate, to compensate for the slow kinetics and competing oxygenation reaction of the key photosynthetic CO2-fixing enzyme rubisco. The limiting CO2-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 CO2-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 ß-CAs. Our results identify the LCIB family as a previously unidentified group of ß-CAs, and provide a biochemical foundation for their function in the microalgal CCMs.
Asunto(s)
Dióxido de Carbono/química , Anhidrasas Carbónicas/química , Chlamydomonas reinhardtii/enzimología , Fotosíntesis , Proteínas de Plantas/química , Aire , Carbono/metabolismo , Dominio Catalítico , Cloroplastos/enzimología , Clonación Molecular , Iones , Microalgas/enzimología , Conformación Molecular , Mutación , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Agua/química , Zinc/químicaRESUMEN
Carbonic anhydrases (CAs) are a well characterized family of metalloenzymes that are highly efficient in facilitating the interconversion between carbon dioxide and bicarbonate. Recently, CA activity has been associated with the LCIB (limiting CO2-inducible protein B) protein family, which has been an interesting target in aquatic photosynthetic microorganisms. To gain further insight into the catalytic mechanism of this new group of CAs, the X-ray structure of a highly active LCIB homolog (PtLCIB3) from the diatom Phaeodactylum tricornutum was determined. The CA activities of PtLCIB3, its paralog PtLCIB4 and a variety of their mutants were also measured. It was discovered that PtLCIB3 has a classic ß-CA fold and its overall structure is highly similar to that of its homolog PtLCIB4. Subtle structural alterations between PtLCIB3 and PtLCIB4 indicate that an alternative proton-shuttle cavity could perhaps be one reason for their remarkable difference in CA activity. A potential alternative proton-shuttle route in the LCIB protein family is suggested based on these results.