Nicotinamide Cytosine Dinucleotide Research Articles

Competitive inhibition of a non-natural cofactor dependent formaldehyde dehydrogenase by imidazole.

To better understand the unique inhibitory behavior of a non-natural cofactor preferred formaldehyde dehydrogenase (FalDH) mutant 9B2. We described our serendipitous observation that 9B2 was reversibly inhibited by residual imidazole introduced during protein preparation, while the wild-type enzyme was not sensitive to imidazole. Kinetic analysis showed that imidazole was a competitive inhibitor of formaldehyde with a Ki of 16 μM and an uncompetitive inhibitor of Nicotinamide Cytosine Dinucleotide for 9B2, indicating that formaldehyde and imidazole were combined in the same position. Molecular docking results of 9B2 showed that imidazole could favorably bind very close to the nicotinamide moiety of the cofactor, where formaldehyde was expected to reside for catalysis, which was in line with a competitive inhibition. The mutant 9B2 can be competitively inhibited by imidazole, suggesting that cautions should be taken to evaluate activities as protein mutants might attain unexpected sensitivity to a component in buffers for purification or activity assays.

Creation of cytochrome P450 catalysis depending on a non-natural cofactor for fatty acid hydroxylation

Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate (NADPH), however, their applications remain limited largely because NADPH is cost-prohibitive for biocatalysis at scale yet tightly regulated in host cells. A highly challenging task for P450 catalysis has been to develop an alternative and biocompatible electron-donating system. Here we engineered P450 BM3 to favor reduced nicotinamide cytosine dinucleotide (NCDH) and created non-natural cofactor-dependent P450 catalysis. Two outstanding mutants were identified with over 640-fold NCDH preference improvement and good catalytic efficiencies of over 15,000 M−1 s−1 for the oxidation of the fatty acid probe 12-(para-nitrophenoxy)-dodecanoate. Molecular docking analysis indicated that these mutants bear a compacted cofactor entrance. Upon fusing with an NCD-dependent formate dehydrogenase, fused proteins functioned as NCDH-specific P450 catalysts by using formate as the electron donor. Importantly, these mutants and fusions catalyzed NCDH-dependent hydroxylation of fatty acids with similar chain length preference to those by natural P450 BM3 in the presence of NADPH and also similar regioselectivity for subterminal hydroxylation of lauric acid. As P450 BM3 and its variants are catalytically powerful to take diverse substrates and convey different reaction paths, our results offer an exciting opportunity to devise advanced cell factories that convey oxidative biocatalysis with an orthogonal reducing power supply system.

A phosphite-based screening platform for identification of enzymes favoring nonnatural cofactors

Enzymes with dedicated cofactor preference are essential for advanced biocatalysis and biomanufacturing, especially when employing nonnatural nicotinamide cofactors in redox reactions. However, directed evolution of an enzyme to switch its cofactor preference is often hindered by the lack of efficient and affordable method for screening as the cofactor per se or the substrate can be prohibitively expensive. Here, we developed a growth-based selection platform to identify nonnatural cofactor-dependent oxidoreductase mutants. The growth of bacteria depended on the nicotinamide cytosine dinucleotide (NCD) mediated conversion of non-metabolizable phosphite into phosphate. The strain BW14329 lacking the ability to oxidize phosphite was suitable as host, and NCD-dependent phosphite dehydrogenase (Pdh*) is essential to the selection platform. Previously confirmed NCD synthetase with NCD synthesis capacity and NCD-dependent malic enzyme were successfully identified by using the platform. The feasibility of this strategy was successfully demonstrated using derived NCD-active malic enzyme as well as for the directed evolution of NCD synthetase in Escherichia coli. A phosphite-based screening platform was built for identification of enzymes favoring nonnatural cofactor NCD. In the future, once Pdh variants favoring other biomimetic or nonnatural cofactors are available this selection platform may be readily redesigned to attain new enzyme variants with anticipated cofactor preference, providing opportunities to further expand the chemical space of redox cofactors in chemical biology and synthetic biology.

Engineering Formaldehyde Dehydrogenase from Pseudomonas putida to Favor Nicotinamide Cytosine Dinucleotide

The enzyme formaldehyde dehydrogenase (FalDH) from Pseudomonas putida is of particular interest for biotechnological applications as it catalyzes the oxidation of formaldehyde independent of glutathione. However, the consumption of a stoichiometric amount of nicotinamide adenine dinucleotide (NAD) can be challenging at the metabolic level as this may affect many other NAD-linked processes. A potential solution is to engineer FalDH to utilize non-natural cofactors. Here we devised FalDH variants to favor nicotinamide cytosine dinucleotide (NCD) by structure-guided modification of the binding pocket for the adenine moiety of NAD. Several mutants were obtained and the best one FalDH 9B2 had over 150-fold higher preference for NCD than NAD. Molecular docking analysis indicated that the cofactor binding pocket shrunk to better fit NCD, a smaller-sized cofactor. FalDH 9B2 together with other NCD-linked enzymes offer opportunities to assemble orthogonal pathways for biological conversion of C1 molecules.

Gram-scale biocatalytic preparation of the non-natural cofactor nicotinamide cytosine dinucleotide

A simple and fast method of preparation of non-natural nicotinamide adenine dinucleotide (NAD) analog nicotinamide cytosine dinucleotide (NCD) was developed. NCD synthetase (NcdS-1) was developed from nicotinic acid mononucleotide adenylyltransferase (NadD) with higher catalytic efficiency towards nicotinamide mononucleotide (NMN) and cytidine triphosphate (CTP). Reaction composition was optimized with inorganic pyrophosphatase (PPase) from E. coli to achieve near quantitative conversion of substrates. A Scalable purification procedure was determined to achieve a fast 4.19 g NCD preparation of 96 % purity.

Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology.

Structural Insights into Malic Enzyme Variants Favoring an Unnatural Redox Cofactor.

The use of nicotinamide cytosine dinucleotide (NCD), a biocompatible nicotinamide adenosine dinucleotide (NAD) analogue, is of great scientific and biotechnological interest. Several redox enzymes have been devised to favor NCD, and have been successfully applied in creating NCD-dependent redox systems. However, molecular interactions between cofactor and protein have still to be disclosed in order to guide further engineering efforts. Here we report the structural analysis of an NCD-favoring malic enzyme (ME) variant derived from Escherichia coli. The X-ray crystal structure data revealed that the residues located at position 346 and 401 in ME acted as the "gatekeepers" of the adenine moiety binding cavity. When Arg346 was substituted with either acidic or aromatic residues, the corresponding mutants showed substantially reduced NCD preference. Inspired by these observations, we generated Lactobacillus helveticus derived d-lactate dehydrogenase variants at Ile177, the counterpart to Arg346 in ME, and found a similar trend in terms of cofactor preference changes. As many NAD-dependent oxidoreductases share key structural features, our results provide guidance for protein engineering to obtain more NCD-favoring variants.

Structure-Guided Design of Formate Dehydrogenase for Regeneration of a Non-Natural Redox Cofactor.

Formate dehydrogenase (FDH) has been widely used for the regeneration of the reduced nicotinamide adenine dinucleotide (NADH). To utilize nicotinamide cytosine dinucleotide (NCD) as a non-natural redox cofactor, it remains challenging as NCDH, the reduced form of NCD, has to be efficiently regenerated. Here we demonstrate successful engineering of FDH for NCDH regeneration. Guided by the structural information of FDH from Pseudomonas sp. 101 (pseFDH) and the NAD-pseFDH complex, semi-rational strategies were applied to design mutant libraries and screen for NCD-linked activity. The most active mutant reached a cofactor preference switch from NAD to NCD by 3700-fold. Homology modeling analysis showed that these mutants had reduced cofactor binding pockets and dedicated hydrophobic interactions for NCD. Efficient regeneration of NCDH was implemented by powering an NCD-dependent D-lactate dehydrogenase for stoichiometric and stereospecific reduction of pyruvate to D-lactate at the expense of formate.

Engineering d-Lactate Dehydrogenase to Favor an Non-natural Cofactor Nicotinamide Cytosine Dinucleotide.

Synthetic nicotinamide adenine dinucleotide (NAD) analogues are of great scientific and biotechnological interest. One such analogue, nicotinamide cytosine dinucleotide (NCD), has been successfully applied to creating bioorthogonal redox systems. Yet, only a few redox enzymes have been devised to favor NCD. We have engineered Lactobacillus helveticus-derived NAD-dependent d-lactate dehydrogenase (LhDLDH) to favor NCD by semirational design. Sequence alignment and structural analysis revealed that amino acid residues I177 and N213 form a "gate" guarding the NAD adenine moiety binding cavity. Saturated mutagenesis libraries were constructed by using the mutant LhDLDH-V152R as the parental sequence. Mutants were obtained with good catalytic efficiency, and NCD preference increased by up to 940-fold. Experiments showed that Escherichia coli cells expressing mutants with higher NCD preference afforded much less d-lactate, thus suggesting the potential to construct NCD-mediated orthogonal metabolism.

Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate

AbstractA non‐natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non‐natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD‐dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO2 source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When 13C‐isotope‐labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom‐economic strategy for CO2 fixation where formate is used in lieu of CO2 and offers dedicated reducing power.

Non-natural Cofactor and Formate-Driven Reductive Carboxylation of Pyruvate.

A non-natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non-natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD-dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO2 source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When 13 C-isotope-labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom-economic strategy for CO2 fixation where formate is used in lieu of CO2 and offers dedicated reducing power.

Escherichia coli Strain Designed for Characterizing in Vivo Functions of Nicotinamide Adenine Dinucleotide Analogues.

An Escherichia coli strain was constructed for the efficient import of nicotinamide adenine dinucleotide (NAD) analogues into cells by limiting extracellular degradation while expressing an efficient NAD importer. In vivo functions of three NAD analogues were characterized. Nicotinamide hypoxanthine dinucleotide was identified as an inhibitor of NAD synthesis. Nicotinamide cytosine dinucleotide had excellent biocompatibility and was used for characterizing a growth-dependent degradation of in vivo nicotinamide cofactors.

Synthetic Cofactor-Linked Metabolic Circuits for Selective Energy Transfer

A cellular energy-transfer process can be analogized as the running of an energy carrier (EC)-linked metabolic circuit between an energy-supplying module (ESM) and an energy-utilizing module (EUM). Because natural EC such as the reduced nicotinamide adenosine dinucleotide (NAD) links multiple energy-transfer modules and metabolic circuits, and the formation of natural EC is routinely coupled with the transformation of endogenous substances, it is challenging to transfer energy selectively. Here we devise synthetic cofactor-linked circuits for pathway-selective energy transfer. We engineer phosphite dehydrogenase as ESM to use the synthetic cofactor nicotinamide cytosine dinucleotide (NCD). We construct diverse circuits in vitro by combining different ESM, EUM, and EC, and we demonstrate that an energy-transfer process is controllable by tuning the feature of each component of the circuit. More specifically, we show that it is possible to drive the NCD-linked subsystem while leaving the NAD-linked reaction...

Creation of Bioorthogonal Redox Systems Depending on Nicotinamide Flucytosine Dinucleotide

Many enzymes catalyzing biological redox chemistry depend on the omnipresent cofactor, nicotinamide adenine dinucleotide (NAD). NAD is also involved in various nonredox processes. It remains challenging to disconnect one particular NAD-dependent reaction from all others. Here we present a bioorthogonal system that catalyzes the oxidative decarboxylation of l-malate with a dedicated abiotic cofactor, nicotinamide flucytosine dinucleotide (NFCD). By screening the multisite saturated mutagenesis libraries of the NAD-dependent malic enzyme (ME), we identified the mutant ME-L310R/Q401C, which showed excellent activity with NFCD, yet marginal activity with NAD. We found that another synthetic cofactor, nicotinamide cytosine dinucleotide (NCD), also displayed similar activity with the ME mutants. Inspired by these observations, we mutated d-lactate dehydrogenase (DLDH) and malate dehydrogenase (MDH) to DLDH-V152R and MDH-L6R, respectively, and both mutants showed fully active with NFCD. When coupled with DLDH-V152R, ME-L310R/Q401C required only a catalytic amount of NFCD to convert l-malate. Our results opened the window to engineer bioorthogonal redox systems for a wide variety of applications in systems biology and synthetic biology.

Zum wirkungsmechanismus von dehydrogenasen

During systematic investigations, the adenine-free derivatives of NAD and 3-acetylpyridine-adenine dinucleotide, 3-acetylpyridine mononucleotide and nicotinamide-cytosine dinucleotide were prepared in a pure state and were characterized. A comparison of the absorption spectra of the dihydroderivatives and of the cyanide and sulphite addition compounds indicated that an interaction took place between both heterocyclic bases. The properties of the NAD models were studied under the same conditions with crystalline yeast and liver alcohol dehydrogenase, rabbit-skeletal-muscle and pig-heart lactic dehydrogenase, mitochondrial horse-heart malic dehydrogenase, ox-liver glutamic dehydrogenase and rabbit-skeletal-muscle glyceraldehyde-3-phosphate dehydrogenase. The adenine-free compounds were reduced in all systems but at greatly different rates. The Michaelis constants of some of the coenzyme models and substrates were determined. The values showed that pyrophosphate and adenine parts were important in the binding as well as in the activation. Im Rahmen systematischer Untersuchungen wurden die adeninfreien Verbindungen von Nicotinamid-adenin-dinucleotid und 3-Acetylpyridin-adenin-dinucleotid, das 3-Acetylpyridin-mononucleotid und Nicotinamid-cytosin-dinucleotid rein dargestellt und charakterisiert. Aus dem Vergleich der Absorptionsspektren der Dihydroverbindungen und der Cyanid- und Sulfit-Additionsverbindungen läßt sich eine Wechselwirkung zwischen den beiden heterocyclischen Basen ableiten. Die Wirksamkeit der Nicotinamid-adenin-dinucleotid-Modelle wurde unter gleichartigen Reaktionsbedingungen an kristallisierter Hefe- und Leber-Alkohol-dehydrogenase, Kaninchenskeletmuskel- und Schweineherz-l-Lactat-dehydrogenase, mitochondrialer Pferdeherz-l-Malat-dehydrogenase, Rinderleber-l-Glutamat-dehydrogenase und Kaninchenskeletmuskel-Glycerin-aldehyd-3-phosphatdehydrogenase studiert. Die adeninfreien Verbindungen wurden von allen Systemen, allerdings mit sehr unterschiedlicher Geschwindigkeit, reduziert. Es wurden einige Michaeliskonstanten von Coenzymmodellen und Substraten bestimmt. Die Werte lassen erkennen, daß Pyrophosphat- und Adeninteile sowohl für die Bindung als auch für die Aktivierung eine Rolle spielen können.