Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and vice versa. Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity.

Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol’s enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.

The development of sustainable processes for the valorization of byproducts and other waste streams remains an ongoing challenge in the field of catalysis. Racemic borneol, isoborneol and camphor are currently produced from α‐pinene, a side product from the production of cellulose. The pure enantiomers of these monoterpenoids have numerous applications in cosmetics and act as reagents for asymmetric synthesis, making an enzymatic route for their separation into optically pure enantiomers a desirable goal. Known short‐chain borneol‐type dehydrogenases (BDHs) from plants and bacteria lack the required specificity, stability or activity for industrial utilization. Prompted by reports on the presence of pure (−)‐borneol and (−)‐camphor in essential oils from rosemary, we set out to investigate dehydrogenases from the genus Salvia and discovered a dehydrogenase with high specificity (E>120) and high specific activity (>0.02 U mg⁻¹) for borneol and isoborneol. Compared to other specific dehydrogenases, the one reported here shows remarkably higher stability, which was exploited to obtain the first three‐dimensional structure of an enantiospecific borneol‐type short‐chain dehydrogenase. This, together with docking studies, led to the identification of a hydrophobic pocket in the enzyme that plays a crucial role in the stereo discrimination of bornane‐type monoterpenoids. The kinetic resolution of borneol and isoborneol can be easily integrated into the existing synthetic route from α‐pinene to camphor thereby allowing the facile synthesis of optically pure monoterpenols from an abundant renewable source.

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial in order to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryoEM) has revolutionized structural biology, its applicability to high-resolution structural analysis of comparatively small enzymes has so far been largely unexplored. Here, it is shown that cryoEM can reveal the structures of plant borneol dehydrogenases of ∼120 kDa at or below 2 Å resolution, paving the way for the rapid development of new biocatalysts that can provide access to bioactive terpenes and terpenoids.

Protein stabilization is a "Holy Grail" of biocatalysis, and stability design is an area of intense research interest. While it is increasingly feasible to effectively increase enzyme thermostability, optimization without compromising activity or selectivity remains a significant challenge. Here, we use full-atom protein sequence design with sidechain conditioning (FAMPNN) to engineer thermostable variants of the borneol dehydrogenase from Salvia rosmarinus (SrBDH1), an enzyme from a family where unselective enzymes dominate, and selectivity is determined by dynamical considerations. By combining FAMPNN design with residue conservation analysis and avoiding active site residues, we were able to computationally design SrBDH1 variants with up to 10 °C enhanced thermostability and strongly increased half-life time at elevated temperature, while retaining selectivity towards (+)-borneol. This design framework, integrating de novo and physics-based protein design tools, demonstrates that stability can be enhanced without disrupting functionally relevant dynamics, providing a route to engineer robust and selective biocatalysts.

Enzyme engineering has produced numerous methods to optimize enzymes for biotechnological processes; however, less is known about how natural evolution creates new functionalities. We investigate the evolutionary emergence of enantioselectivity in plant borneol dehydrogenases (BDHs), which feature hydrophobic active-sites and are enantioselective towards dibornane-type monoterpenols. Ancestral sequence reconstruction provided a trajectory from the oldest unselective BDH ancestor N30 (E=12) toward a more recent selective ancestor N32, involving 19 mutations: 18 mutations are peripheral, one (I111L) occurs in the active-site. The mutation L111I in the hydrophobic pocket increased the selectivity of N30, while the back-mutation I111L decreased the selectivity of N32. Additional peripheral mutations (V136L/G169A/V183I) were required for high selectivity. Crystal structures suggested that protein dynamics, rather than structural changes shape these catalytic properties; this was confirmed by ML/MM simulations of ligand binding. Funnel-metadynamics simulations revealed a correlation between the active-site’s solvent-accessible surface area (SASA) and selectivity. This potential evolutionary pathway shapes enantioselectivity, and guides future enzyme engineering campaigns.