RESUMEN
Carbonic anhydrases (CAs) are extremely fast enzymes, which have attracted much interest in the past due to their medical relevance and their biotechnological potential. An α-type CA gene was isolated from DNA derived from an active hydrothermal vent chimney, in an effort to identify novel CAs with suitable properties for CO2 capture. The gene product was recombinantly produced and characterized, revealing remarkable thermostability, also in the presence of high ionic strength alkaline conditions, which are used in some CO2 capture applications. The Tm was above 90⯰C under all tested conditions. The enzyme was crystallized and the structure determined by molecular replacement, revealing a typical bacterial α-type CA non-covalent dimer, but not the disulphide mediated tetramer observed for the hyperthermophilic homologue used for molecular replacement, from Thermovibrio ammonificans. Structural comparison suggests that an increased secondary structure content, increased content of charges on the surface and ionic interactions compared to mesophilic enzymes, may be main structural sources of thermostability, as previously suggested for the homologue from Sulfurihydrogenibium yellowstonense.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/química , Anhidrasas Carbónicas/química , Respiraderos Hidrotermales/microbiología , Secuencia de Aminoácidos , Bacterias/genética , Bacterias/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Dimerización , Estabilidad de Enzimas , Calor , Concentración de Iones de Hidrógeno , Cinética , Metagenoma , Alineación de SecuenciaRESUMEN
The crystal structure of Aspergillus oryzae carbonic anhydrase (AoCA) was determined at 2.7Å resolution and it revealed a dimer, which only has precedents in the α class in two membrane and cancer-associated enzymes. α carbonic anhydrases are underrepresented in fungi compared to the ß class, this being the first structural representative. The overall fold and zinc binding site resemble other well studied carbonic anhydrases. A major difference is that the histidine, thought to be the major proton shuttle residue in most mammalian enzymes, is replaced by a phenylalanine in AoCA. This finding poses intriguing questions as to the biological functions of fungal α carbonic anhydrases, which are promising candidates for biotechnological applications.
