Rational Design of Thermostable Carbonic Anhydrase Mutants Using Molecular Dynamics Simulations

Abstract

The stability of enzymes is critical for their application in industrial processes, which generally require different conditions from the natural enzyme environment. Both rational and random protein engineering approaches have been used to increase stability, with the latter requiring extensive experimental effort for the screening of variants. Moreover, some general rules addressing the molecular origin of protein thermostability have been established. Herein, we demonstrate the use of molecular dynamics simulations to gain molecular level understanding of protein thermostability and to engineer stabilizing mutations. Carbonic anhydrase (CA) is an enzyme with a high potential for biotechnological carbon capture applications, provided it can be engineered to withstand the high temperature process environments, inevitable in most gas treatment units. In this study, we used molecular dynamics simulations at 343, 353, and 363 K to study the relationship between structure flexibility and thermostability in bacterial α-CAs and applied this knowledge to the design of mutants with increased stability. The most thermostable α-CA known, TaCA from Thermovibrio ammonificans, had the most rigid structure during molecular dynamics simulations, but also showed regions with high flexibility. The most flexible amino acids in these regions were identified from root mean square fluctuation (RMSF) studies, and stabilizing point mutations were predicted based on their capacity to improve the calculated free energy of unfolding. Disulfide bonds were also designed at sites with suitable geometries and selected based on their location at flexible sites, assessed by B-factor calculation. Molecular dynamics simulations allowed the identification of five mutants with lower RMSF of the overall structure at 400 K, compared to wild-type TaCA. Comparison of free-energy landscapes between wild-type TaCA and the most promising mutants, Pro165Cys-Gln170Cys and Asn140Gly, showed an increased conformational stability of the mutants at 400 K.

Enhanced Thermostability and Enzymatic Activity of cel6A Variants from Thermobifida fusca by Empirical Domain Engineering

“…To analyze denaturation process in cel6A constructs within the reasonable time limits, much higher temperatures are used. MD simulations procedures have been performed at higher temperatures previously to study the thermostability in various enzymes [ 15 , 36 , 38 , 69 , 70 ]. To investigate the global structural stability of cel6A.CBC, a comparative molecular dynamics (MD) simulation, was carried out at different temperatures.…”

“…MD simulations (molecular dynamics, is used to analyze physical movements of atoms and molecules by using computational tools) have been used in several studies to evaluate the factors regulating thermostability of enzymes [ 11 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ] and to investigate the stability of the biomolecular complex and interaction attributes [ 31 , 32 ]. These include the thermal stable mechanisms of rubredoxin [ 33 ], nuclease [ 34 ], barnase [ 35 ], nitrile hydratase [ 36 ], adenylate kinase [ 37 ], carbonic anhydrase [ 38 ], carboxylesterase from Geobacillus stearothermophilus [ 39 ], psychrophilic esterase from Pseudoalteromonas haloplanktis [ 40 ], hyperthermophilic esterase from Archaeoglobus fulgidus [ 41 ], thermostable para-nitrobenzyl esterase from Bacillus subtilis [ 42 ], and CBMs from Clostridium cellulovorans [ 43 , 44 ].…”

Conformational Dynamics‐Guided Loop Engineering of an Alcohol Dehydrogenase: Capture, Turnover and Enantioselective Transformation of Difficult‐to‐Reduce Ketones

“…Loop motion and flexibility at the binding pocket play crucial roles in the optimal functions of enzymes . Induced fluctuation by introducing appropriate mutations lead to loop movements that can reshape the binding pocket, and therefore influence substrate capture and turnover.…”

“…In recent years, there has been growing interest in the conformational dynamics of proteins, which play an important role in enzyme catalysis, and engineering of enzymes guided by conformational dynamics has become an effective strategy for protein evolution, achieving successes in expanding substrate scope, increasing enantioselectivity, relieving product inhibition, and improving thermostability . The motion of amino acid residues in loops involved in the pocket has been recognized to influence the catalytic properties of enzymes .…”

Characterization and High-Level Periplasmic Expression of Thermostable α-Carbonic Anhydrase from Thermosulfurimonas Dismutans in Escherichia Coli for CO2 Capture and Utilization

“…CAs from thermophiles, halophiles, and alkaliphiles have been searched and examined for their potential industrial applicability [9][10][11][12][13][14][15]. Some researchers have shown that the stability of CA can be improved by protein engineering via rational design [16][17][18][19][20] or directed evolution [21]. However, the stabilities of CAs were not sufficiently high in most cases, and high stabilities were achieved only under specific test conditions such as high-salt condition [12,18] or organic solvent [21].…”