Structure of a hyperthermostable carbonic anhydrase identified from an active hydrothermal vent chimney

Abstract

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 CO 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 CO capture applications. The T 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.

Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1

“…Diffraction data were processed using the HKL2000 package (HKL Research, USA). The pm CA structure was determined by molecular replacement (Phaser, CCP4) using the α-CA structure from the Logatchev hydrothermal field metagenome (LOGACA, PDB code 6EKI) as a search model ( Fredslund et al, 2018 ). Refinement of the structure was performed using REFMAC5 and PHENIX, with non-crystallographic symmetry and secondary structure restraints ( Collaborative Computational Project, Number 4, 1994 ).…”

Alpha-Carbonic Anhydrases from Hydrothermal Vent Sources as Potential Carbon Dioxide Sequestration Agents: In Silico Sequence, Structure and Dynamics Analyses

“…This is in contrast to the β-CA and γ-CA classes that are found in the cytoplasm [14]. Previously isolated bacterial α-CAs, including LOGACA, PmCA and TaCA, possess signal peptides at the N-terminal of the protein [29,35,85]. In this study, probable cleavage sites for the retrieved sequences were calculated by two signal peptide prediction programs.…”

“…They also have a hydrophobic pocket close to the active site where the CO 2 molecule is normally held during catalysis (Figure 1D). This pocket has been identified in most α-CAs, including the human CA II [33][34][35][36][37].…”

“…This has been observed in vitro on α-CAs from hydrothermal vent bacteria Caminibacter mediatlanticus (CmCA), Persephonella marina (PmCA) and TaCA, with CmCA and PmCA revealing stability up to 70 • C and 100 • C, respectively, and the wild-type and variants of TaCA appearing stable up to 95 • C [38][39][40][41]. Another CA, found in previously isolated DNA from the Logatchev hydrothermal field (LOGACA), has also proven to be thermostable, withstanding temperatures up to 103 • C [35]. The structures of these α-CAs, except CmCA, have been experimentally solved and reported [29,35,42].…”

“…Another CA, found in previously isolated DNA from the Logatchev hydrothermal field (LOGACA), has also proven to be thermostable, withstanding temperatures up to 103 • C [35]. The structures of these α-CAs, except CmCA, have been experimentally solved and reported [29,35,42]. Consequently, the in silico analysis of the thermostability attributes of α-CAs from hydrothermal vents, is the focus of this study.…”