Structural and catalytic effects of proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase II

Abstract

The bioengineering of a thermophilic enzyme starting from a mesophilic scaffold has proven to be a significant challenge as several stabilizing elements have been proposed to be the foundation of thermal stability including disulfide bridges, surface loop reduction, ionic pair networks, proline substitutions, and aromatic clusters. This study emphasizes the impact of increasing the rigidity of human carbonic anhydrase II (HCA II) via incorporation of proline residues at positions 170 and 234, which are located in surface loops that are able to accommodate restrictive main-chain conformations without rearrangement of the surrounding peptide backbone. Additionally, the effect of compactness of HCA II was examined by way of deletion of a surface loop (residues 230 through 240), which had been previously identified as a possible source of thermal stability for the hyperthermophilic CA isolated from the bacterium Sulfurihydrogenibium yellowstonense YO3AOP1. Differential scanning calorimetry analysis of these HCA II variants revealed that these structural modifications had a minimum effect on the thermal stability of the enzyme while kinetic studies showed unexpected effects on the catalytic efficiency and proton transfer rates. X-ray crystallographic analysis of these HCA II variants showed the electrostatic potential and configuration of the highly acidic loop (residues 230 and 240) plays an important role in its high catalytic activity. Based on these observations and the literature, a picture is emerging of the various components within the general structural architecture of HCA II that are key to stability. These elements could provide the blueprints for the rational thermal stability engineering of other enzymes.

Structural and biophysical characterization of the α-carbonic anhydrase from the gammaproteobacteriumThiomicrospira crunogenaXCL-2: insights into engineering thermostable enzymes for CO2sequestration

“…S5). The compact structure, the presence of an intramolecular disulfide bond and the dimeric arrangement of TcruCA are proposed to contribute significantly to the thermostability of the enzyme (Boone, Habibzadegan, Tu et al, 2013;Boone et al, 2015). Similar to other -CAs, the active site of TcruCA is subdivided into a hydrophilic and a hydrophobic pocket (Fig.…”

“…As such, TcruCA exhibits a reduced catalytic efficiency (approximately tenfold) compared with hCA II, which is most likely attributable to these residue substitutions (Table 3). Furthermore, the reduced catalytic efficiency might also be a result of the loop deletion in region 1 (corresponding to residues 230-240 in hCA II), which has recently been suggested to be important for catalysis (Boone et al, 2015).…”

“…These criteria alone would potentially make TcruCA, or any other dimeric -CA, a good candidate for industrial use owing to its attributed thermostability. To date, protein engineering of a thermostable hCA II variant has been focused on alterations in the monomeric structure of the enzyme (Boone, Habibzadegan, Gill et al, 2013;Boone, Habibzadegan, Tu et al, 2013;Boone et al, 2015). Although this has been successful in increasing the overall conformational stability of the enzyme, none of these variants have been utilized for industrial applications.…”

“…Another possibility for the decreased k cat /K M is a loss of the region 1 loop (residues 230-240 in hCA II), which has been associated with a reduction in catalytic efficiency (Boone et al, 2015). In hCA II this region contains four Glu residues, which may establish an internal buffering mechanism, similar to the predicted function of the proteoglycan domain of hCA IX (Alterio et al, 2009), which may enhance substrate/product entry/exit from the active site.…”

Engineering the thermostability of β-glucuronidase from Penicillium purpurogenum Li-3 by loop transplant

“…This property often results in the high flexibility of loops which is primarily responsible for the instability of protein (Vemparala et al 2011). Therefore, manipulating the dynamic loops such as truncation may be expected to enhance the enzyme stability (Boone et al 2015). A few successful examples have also been reported in literature, for example, Damnjanovic et al deleted a surface loop 38-46 of phospholipase D, and the half-life at 70°C increased by 11.7 time compared to wild-type (Damnjanovic et al 2014).…”