Abstract:Coenzyme engineering, especially for altered coenzyme specificity, has been a research hotspot for more than a decade. In the present study, a novel computational strategy that enhances the hydrogen-bond interaction between an enzyme and a coenzyme was developed and utilized to alter the coenzyme preference. This novel computational strategy only required the structure of the target enzyme. No other homologous enzymes were needed to achieve alteration in the coenzyme preference of a certain enzyme. Using our n… Show more
“…Therefore, in a protein-ligand biosystem, the halogen bond can shift the enzyme’s substrate selectivity by adjusting the substrate binding conformation. It can be used as a powerful tool, similarly to hydrogen bond 7 8 31 32 and electrostatic interactions 33 in enzyme design. Consequently, the halogen bond in the biocatalysis cannot be ignored and more attention should be paid to it.…”
The use of halogen bond is widespread in drug discovery, design, and clinical trials, but is overlooked in drug biosynthesis. Here, the role of halogen bond in the nitrilase-catalyzed synthesis of ortho-, meta-, and para-chlorophenylacetic acid was investigated. Different distributions of halogen bond induced changes of substrate binding conformation and affected substrate selectivity. By engineering the halogen interaction, the substrate selectivity of the enzyme changed, with the implication that halogen bond plays an important role in biosynthesis and should be used as an efficient and reliable tool in enzymatic drug synthesis.
“…Therefore, in a protein-ligand biosystem, the halogen bond can shift the enzyme’s substrate selectivity by adjusting the substrate binding conformation. It can be used as a powerful tool, similarly to hydrogen bond 7 8 31 32 and electrostatic interactions 33 in enzyme design. Consequently, the halogen bond in the biocatalysis cannot be ignored and more attention should be paid to it.…”
The use of halogen bond is widespread in drug discovery, design, and clinical trials, but is overlooked in drug biosynthesis. Here, the role of halogen bond in the nitrilase-catalyzed synthesis of ortho-, meta-, and para-chlorophenylacetic acid was investigated. Different distributions of halogen bond induced changes of substrate binding conformation and affected substrate selectivity. By engineering the halogen interaction, the substrate selectivity of the enzyme changed, with the implication that halogen bond plays an important role in biosynthesis and should be used as an efficient and reliable tool in enzymatic drug synthesis.
“…This novel computational strategy only required the structure of the target enzyme without other homologous enzymes. By using this rational design method, Gluconobacter oxydans Gox2181, which belongs to the short-chain dehydrogenases/reductases superfamily (SDR superfamily), was engineered to show a much higher enzymatic activity with NADPH as its coenzyme through the two-site mutation of Q20R and D43S [67] . Fig.…”
Section: Coenzyme Engineering Methods Of Nicotinamide-based Coenzymesmentioning
Two natural nicotinamide-based coenzymes (NAD and NADP) are indispensably required by the vast majority of oxidoreductases for catabolism and anabolism, respectively. Most NAD(P)-dependent oxidoreductases prefer one coenzyme as an electron acceptor or donor to the other depending on their different metabolic roles. This coenzyme preference associated with coenzyme imbalance presents some challenges for the construction of high-efficiency in vivo and in vitro synthetic biology pathways. Changing the coenzyme preference of NAD(P)-dependent oxidoreductases is an important area of protein engineering, which is closely related to product-oriented synthetic biology projects. This review focuses on the methodology of nicotinamide-based coenzyme engineering, with its application in improving product yields and decreasing production costs. Biomimetic nicotinamide-containing coenzymes have been proposed to replace natural coenzymes because they are more stable and less costly than natural coenzymes. Recent advances in the switching of coenzyme preference from natural to biomimetic coenzymes are also covered in this review. Engineering coenzyme preferences from natural to biomimetic coenzymes has become an important direction for coenzyme engineering, especially for in vitro synthetic pathways and in vivo bioorthogonal redox pathways.
“…Furthermore, the catalytic efficiency and coenzyme specificity is largely dictated by the charge and polarity of key active site residues 43 . Only a few studies used MD simulations to understand these interactions 44 . In our work, MD simulations highlighted the important role of the mutations in PseFDH V9…”
Section: Molecular Basis Of Psefdh V9 Specificity Towards Nadp +mentioning
CGAR) 2 A b s t r a c t Efficient regeneration of cofactors is vital for the establishment of continuous biocatalytic processes.Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO 2 ). However, formate dehydrogenases (FDHs) are usual specific to NAD + , such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP + , but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP + , specificity towards NADP + , and affinity towards formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits no initial activity towards NADP + , we generate a library of >10 6 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 13-fold higher specificity than the best enzyme previously engineered, while retaining high affinity towards formate.By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how non-additive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify superior enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.3 I n t r o d u c t i o n Co-factor regeneration is vital for the continuous operation of biocatalytic processes taking place either within a living cell or in a cell free system 1 . A considerable amount of research has therefore been invested in developing and optimizing enzymatic systems for the in situ regeneration of key cofactors such as ATP, NADH, and NADPH 2,3 . Formate has been long considered as a suitable reducing agent for the regeneration of NADH both in vivo and in vitro 2,4 . This is due to several properties: (i) abundance of formate dehydrogenases (FDHs) that can efficiently transfer reducing power from formate to NAD + 5 ; (ii) formate oxidation is practically irreversible, increasing the efficiency of NADH regeneration; (iii) formate, a small molecule, can easily cross membranes, thus being accessible within cellular compartments; and (iv) the byproduct of formate oxidation, CO 2 , is non-toxic and can be easily expelled from the system.Many biocatalytic processes rely on NADPH rather than NADH 6, 7 . Therefore, in the last 20 years multiple studies aimed at identifying FDHs that can naturally accept NADP + or engineering NAD-dependent FDHs to accept the phosphorylated cofactor [8][9][10][11][12][13][14][15][16] . Whi...
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