A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions.

Engineering dual-function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR2 E2 , with efficient native transaminase (kcat : 69.49±1.77 min-1 ) and artificial esterase (kcat : 3908-0.41 min-1 ) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR2 E2 (pHopt : 8.0-9.5; Topt : 60-65 °C) efficiently converts methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate into 3-(R)-amino-4-(2,4,5-trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the ß-keto ester into the ß-keto acid at the hydrolytic site and subsequently into the ß-amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TR2 E2 PluriZyme was proven with a set of ß-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.

Rational Engineering of Multiple Active Sites in an Ester Hydrolase.

Effects of altering the properties of an active site in an enzymatic homogeneous catalyst have been extensively reported. However, the possibility of increasing the number of such sites, as commonly done in heterogeneous catalytic materials, remains unexplored, particularly because those have to accommodate appropriate residues in specific configurations. This possibility was investigated by using a serine ester hydrolase as the target enzyme. By using the Protein Energy Landscape Exploration software, which maps ligand diffusion and binding, we found a potential binding pocket capable of holding an extra catalytic triad and oxyanion hole contacts. By introducing two mutations, this binding pocket became a catalytic site. Its substrate specificity, substrate preference, and catalytic activity were different from those of the native site of the wild type ester hydrolase and other hydrolases, due to the differences in the active site architecture. Converting the binding pocket into an extra catalytic active site was proven to be a successful approach to create a serine ester hydrolase with two functional reactive groups. Our results illustrate the accuracy and predictive nature of modern modeling techniques, opening novel catalytic opportunities coming from the presence of different catalytic environments in single enzymes.

Mapping enzyme-substrate interactions: its potential to study the mechanism of enzymes.

With the increase of the need to use more sustainable processes for the industry in our society, the modeling of enzymes has become crucial to fully comprehend their mechanism of action and use this knowledge to enhance and design their properties. A lot of methods to study enzymes computationally exist and they have been classified on sequence-based, structure-based, and the more new artificial intelligence-based ones. Albeit the abundance of methods to help predict the function of an enzyme, molecular modeling is crucial when trying to understand the enzyme mechanism, as they aim to correlate atomistic information with experimental data. Among them, methods that simulate the system dynamics at a molecular mechanics level of theory (classical force fields) have shown to offer a comprehensive study. In this book chapter, we will analyze these techniques, emphasizing the importance of precise modeling of enzyme-substrate interactions. In the end, a brief explanation of the transference of the information from research studies to the industry is given accompanied with two examples of family enzymes where their modeling has helped their exploitation.

Structural and biochemical insights into an engineered high-redox potential laccase overproduced in Aspergillus.

Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH. SAXS showed both enzymes form monomers in solution, 7D5 laccase with a more oblate geometric structure due to heavier and more heterogeneous glycosylation. The enzyme presents superior catalytic constants towards all tested substrates, with no significant change in optimal pH or redox potential. It shows noticeable high catalytic efficiency with ABTS and dimethyl-4-phenylenediamine, 7 and 32 times better than the wild type, respectively. Computational simulations demonstrated a more favorable binding and electron transfer from the substrate to the T1 copper due to the introduced mutations. PM1 laccase is exceptionally stable to thermal inactivation (t1/2 70 °C = 1.2 h). Yet, both enzymes display outstanding structural robustness at high temperature. They keep folded during 2 h at 100 °C though, thereafter, 7D5 laccase unfolds faster. Rigidification of certain loops due to the mutations added on the protein surface would diminish the capability to absorb temperature fluctuations leading to earlier protein unfolding.

Determinants and Prediction of Esterase Substrate Promiscuity Patterns.

Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.