Essential functional molecules associated with SARS-CoV-2 infection: Potential therapeutic targets for COVID-19.

The whole world is still suffering substantially from the coronavirus disease 2019 (COVID-19) outbreak. Several protein-based molecules that are associated with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which are essential for its functionality, survival, and pathogenesis have been identified and are considered as potential therapeutic targets. These protein-based molecules are either structural/non-structural components of SARS-CoV-2 or host factors, which play a crucial role in this infection. Developing drug molecules against these essential functional molecules to hinder their regular functioning and associated physiological pathways could be promising for successful clinical management of this novel coronavirus infection. The review aims to highlight the functional molecules that play crucial roles in SARS-CoV-2 pathogenesis. We have emphasized how these potential druggable targets could be beneficial in tackling the COVID-19 crisis.

Role of an N-terminal extension in stability and catalytic activity of a hyperthermostable α/ß hydrolase fold esterase.

The carbohydrate esterase family 7 (CE7) enzymes catalyze the deacetylation of acetyl esters of a broad range of alcohols and is unique in its activity towards cephalosporin C. The CE7 fold contains a conserved N-terminal extension that distinguishes it from the canonical α/ß hydrolase fold. The hexameric quaternary structure indicates that the N-terminus may affect activity and specificity by controlling access of substrates to the buried active sites via an entrance tunnel. In this context, we characterized the catalytic parameters, conformation and thermal stability of two truncation variants lacking four and ten residues of the N-terminal region of the hyperthermostable Thermotoga maritima CE7 acetyl esterase (TmAcE). The truncations did not affect the secondary structure or the fold but modulated the oligomerization dynamics. A modest increase was observed in substrate specificity for acetylated xylose compared with acetylated glucose. A drastic reduction of ~30-40°C in the optimum temperature for activity of the variants indicated lower thermal stability. The loss of hyperthermostability appears to be an indirect effect associated with an increase in the conformational flexibility of an otherwise rigid neighboring loop containing a catalytic triad residue. The results suggest that the N-terminal extension was evolutionarily selected to preserve the stability of the enzyme.

Structural role of a conserved active site cis proline in the Thermotoga maritima acetyl esterase from the carbohydrate esterase family 7.

A conserved cis proline residue located in the active site of Thermotoga maritima acetyl esterase (TmAcE) from the carbohydrate esterase family 7 (CE7) has been substituted by alanine. The residue was known to play a crucial role in determining the catalytic properties of the enzyme. To elucidate the structural role of the residue, the crystal structure of the Pro228Ala variant (TmAcEP228A ) was determined at 2.1 Å resolution. The replacement does not affect the overall secondary, tertiary, and quaternary structures and moderately decreases the thermal stability. However, the wild type cis conformation of the 227-228 peptide bond adopts a trans conformation in the variant. Other conformational changes in the tertiary structure are restricted to residues 222-226, preceding this peptide bond and are located away from the active site. Overall, the results suggest that the conserved proline residue is responsible for the cis conformation of the peptide and shapes the geometry of the active site. Elimination of the pyrrolidine ring results in the loss of van der Waals and hydrophobic interactions with both the alcohol and acyl moeities of the ester substrate, leading to significant impairment of the activity and perturbation of substrate specificity. Furthermore, a cis-to-trans conformational change arising out of residue changes at this position may be associated with the evolution of divergent activity, specificity, and stability properties of members constituting the CE7 family. Proteins 2017; 85:694-708. © 2016 Wiley Periodicals, Inc.

Crystal structure of Thermotoga maritima acetyl esterase complex with a substrate analog: Insights into the distinctive substrate specificity in the CE7 carbohydrate esterase family.

The carbohydrate esterase family 7 (CE7) members are acetyl esterases that possess unusual substrate specificity for cephalosporin C and 7-amino-cephalosporanic acid. This family containing the α/ß hydrolase fold has a distinctive substrate profile that allows it to carry out hydrolysis of esters containing diverse alcohol moieties while maintaining narrow specificity for an acetate ester. Here we investigate the structural basis of this preference for small acyl groups using the crystal structure of the thermostable Thermotoga maritima CE7 acetyl esterase (TmAcE) complexed with a non-cognate substrate analog. The structure determined at 1.86 Å resolution provides direct evidence for the location of the largely hydrophobic and rigid substrate binding pocket in this family. Furthermore, a three-helix insertion domain near the catalytic machinery shapes the substrate binding site. The structure reveals two residues (Pro228 and Ile276) which constitute a hydrophobic rigid binding surface for the acyl group of the ester and thus restricts the size of the acyl group that be accommodated. In combination with previous literature on kinetic properties of the enzyme, our studies suggest that these residues determine the unique specificity of the TmAcE for short straight chain esters. The structure provides a template for focused attempts to engineer the CE7 enzymes for enhanced stability, selectivity or activity for biocatalytic applications.

An extended loop in CE7 carbohydrate esterase family is dispensable for oligomerization but required for activity and thermostability.

The carbohydrate esterase family 7 (CE7) belonging to the α/ß hydrolase superfamily contains a structurally conserved loop extension element relative to the canonical α/ß hydrolase fold. This element called the ß-interface loop contributes 20-30% of the total buried surface area at intersubunit interfaces of the functional hexameric state. To test whether this loop is an enabling region for the structure and function of the oligomeric assembly, we designed a truncation variant of the thermostable CE7 acetyl esterase from Thermotoga maritima (TmAcE). Although deletion of 26 out of 40 residues in the loop had little impact on the hexamer formation, the variant exhibited altered dynamics of the oligomeric assembly and a loss of thermal stability. Furthermore, the mutant lacked catalytic activity. Crystal structures of the variant and a new crystal form of the wild type protein determined at 2.75Å and 1.76Å, respectively, provide a rationale for the properties of the variant. The hexameric assembly in the variant is identical to that of the wild type and differed only in the lack of buried surface area interactions at the original intersubunit interfaces. This is accompanied by disorder in an extended region of the truncated loop that consequently induces disorder in the neighboring oxyanion hole loop. Overall, the results suggest that the ß-interface loop in CE7 enzymes is dispensable for the oligomeric assembly. Rather, the loop extension event was evolutionarily selected to regulate activity, conformational flexibility and thermal stability.

Understanding and Optimizing the SMX Static Mixer.

Using the Mapping Method different designs of SMX motionless mixers are analyzed and optimized. The three design parameters that constitute a specific SMX design are: The number of cross-bars over the width of channel, N(x) , the number of parallel cross-bars per element, N(p) , and the angle between opposite cross-bars θ. Optimizing N(x) , somewhat surprisingly reveals that in the standard design with N(p) = 3, N(x) = 6 is the optimum using both energy efficiency as well as compactness as criteria. Increasing N(x) results in under-stretching and decreasing N(x) leads to over-stretching of the interface. Increasing N(p) makes interfacial stretching more effective by co-operating vortices. Comparing realized to optimal stretching, we find the optimum series for all possible SMX(n) designs to obey the universal design rule N(p) = (2/3) N(x) -1, for N(x) = 3, 6, 9, 12, ….