Structure and Cooperativity in Substrate–Enzyme Interactions: Perspectives on Enzyme Engineering and Inhibitor Design

Abstract

Enzyme-based synthetic chemistry provides a green way to synthesize industrially important chemical scaffolds and provides incomparable substrate specificity and unmatched stereo-, regio-, and chemoselective product formation. However, using biocatalysts at an industrial scale has its challenges, like their narrow substrate scope, limited stability in large-scale one-pot reactions, and low expression levels. These limitations can be overcome by engineering and fine-tuning these biocatalysts using advanced protein engineering methods. A detailed understanding of the enzyme structure and catalytic mechanism and its structure− function relationship, cooperativity in binding of substrates, and dynamics of substrate−enzyme−cofactor complexes is essential for rational enzyme engineering for a specific purpose. This Review covers all these aspects along with an in-depth categorization of various industrially and pharmaceutically crucial bisubstrate enzymes based on their reaction mechanisms and their active site and substrate/cofactor-binding site structures. As the bisubstrate enzymes constitute around 60% of the known industrially important enzymes, studying their mechanism of actions and structure−activity relationship gives significant insight into deciding the targets for protein engineering for developing industrial biocatalysts. Thus, this Review is focused on providing a comprehensive knowledge of the bisubstrate enzymes' structure, their mechanisms, and protein engineering approaches to develop them into industrial biocatalysts.

Dissecting the Enantioselective Neurotoxicity of Isocarbophos: Chiral Insight from Cellular, Molecular, and Computational Investigations

“…In fact, almost all the processes of life activity are continuously changing in organisms, and the inquisition of these physiological and pathological phenomena from a dynamic scale can unveil their intrinsic rules. , Meanwhile, the general opinions believe that biomolecular functions depend mainly on its conformations, or that conformational changes determine its biological functions . Moreover, biomacromolecular conformations are not completely rigid in the body; instead it usually has a high degree of inherent flexibility, which has been proved by some experimental techniques such as nuclear magnetic resonance, computational chemistry, and X-ray diffraction crystallography. , However, the physiological significance of biomolecular conformational flexibility has not yet been fully clarified.…”

Expanding the Substrate Scope of Native Amine Dehydrogenases through In Silico Structural Exploration and Targeted Protein Engineering

“…In addition to X-ray crystallography, MD simulations were run to understand the influence of W145A mutation on protein flexibility and specificity towards cofactors and substrates. [8,23] Accordingly, the study was carried out on an extensive set of Enzyme-Cofactor-Ligand systems where both CfusAmDHÀ W145A (M) or CfusAmDH (WT) were considered, in complex with NAD + (NAD) or NADP + (NADP) cofactors, and 2 a (PEN) or 2 c (HEP) ligands.…”

Cover Feature: Expanding the Substrate Scope of Native Amine Dehydrogenases through In Silico Structural Exploration and Targeted Protein Engineering (ChemCatChem 22/2022)

“…In addition to X-ray crystallography, MD simulations were run to understand the influence of W145A mutation on protein flexibility and specificity towards cofactors and substrates. [8,23] Accordingly, the study was carried out on an extensive set of Enzyme-Cofactor-Ligand systems where both CfusAmDHÀW145A (M) or CfusAmDH (WT) were considered, in complex with NAD + (NAD) or NADP + (NADP) cofactors, and 2 a (PEN) or 2 c (HEP) ligands.…”