Biomimetic Cofactors Research Articles

Engineering Glucose Dehydrogenase to Favor Totally Synthetic Biomimetic Cofactors Containing Carboxyl Group.

The utilization of unnatural nicotinamide cofactors for reactions catalyzed by oxidoreductases has gained increasing interest. Totally synthetic nicotinamide cofactor biomimetics (NCBs) are cost-effective and convenient to synthesize. Thus, it has become increasingly important to develop enzymes that accept NCBs. Here, we have engineered SsGDH to favor a newly synthesized unnatural cofactor 3-carbamoyl-1-(4-carboxybenzyl) pyridin-1-ium (BANA+ ). Using in situ ligand minimization tool, sites 44 and 114 were identified as hotspots for mutagenesis. All the double mutants demonstrated 2.7-7.7-fold improvements in catalytic activity, and the best double mutant E44D/E114 L exhibited 10.6-fold increased catalytic efficiency toward BANA+ . These results provide valuable information for the rational engineering of oxidoreductases with versatile NCBs-dependency, as well as the design of novel biomimetic cofactors.

Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor

Enzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduction reaction of NAD+ with androsterone. Based on the structure and catalytic mechanism, we mutated the residues of T11, I13, D41, A70, and I112 and they interacted with different portions of NAD+ to switch cofactor specificity to biomimetic cofactor nicotinamide mononucleotide (NMN+). Compared to wild-type 3α-HSD/CR, the catalytic efficiency of these mutants for NAD+ decreased significantly except for the T11 mutants but changed slightly for NMN+ except for the A70K mutant. The A70K mutant increased the catalytic efficiency for NMN+ by 8.7-fold, concomitant with a significant decrease in NAD+ by 1.4 × 104-fold, resulting in 9.6 × 104-fold cofactor specificity switch toward NMN+ over NAD+. Meanwhile, the I112K variant increased the thermal stability and changed to a three-state transition from a two-state transition of thermal unfolding of wild-type 3α-HSD/CR by differential scanning fluorimetry. Molecular docking analysis indicated that mutations on these residues affect the position and conformation of the docked NAD+ and NMN+, thereby affecting their activity. A70K variant sterically blocks the binding with NAD+, restores the H-bonding interactions of catalytic residues of Y155 and K159 with NMN+, and enhances the catalytic efficiency for NMN+.

Optimization of operation conditions for improved cytochrome P450BM3 enzymatic reaction yield

AbstractThe P450 cytochrome monooxygenase CYP102A1 from Bacillus megaterium, better known as P450BM3, is a heme‐thiolate enzyme that catalyzes the hydroxylation of numerous substrates. Many of the resulting products are of commercial interest to the pharmaceutical and fine chemical industries. Unlike most other P450 cytochromes, P450BM3 is both soluble and fused to its natural redox partner, which supplies the necessary electrons from NADPH to drive its reaction forward. However, the industrial use of this enzyme is limited by its poor stability and its expensive cofactor. In this work, we explore the effects of buffer formulation and temperature on the stability of wildtype and P450BM3 mutant R966D/W1046S as well as on the stability of nicotinamide cofactors NADPH, NADH, and the biomimetic cofactor N‐benzyl‐1,4‐dihydronicotinamide. We demonstrate that cofactor stability is more important to increase product yield than that of the enzyme. We also demonstrate that low temperatures enhance oxido‐reduction reactions coupling, thus resulting in an increase in the molar ratio of p‐nitrophenolate produced from 10‐pNCA per oxidized cofactor. Overall, the optimized reaction conditions lead to a 2 to 2.6‐fold increase in total product output when wildtype P450BM3 or R966D/W1046S mutant is used with either of these three aforementioned cofactors.

DFT Mechanism Studies: Biomimetic 1,4-NADH Chemoselective, Co-factor Regeneration with [Cp*Rh(bpy)H]+, in Tandem with the Biocatalysis Pathways of a Core Model of the (HLADH)-Zn(II) Mediated Enzyme, in the Enantioselective Reduction of Achiral Ketones to Chiral S-Alcohols

Quantum Chemical (QC) calculations, utilizing Density Functional Theory (DFT), were performed to investigate the mechanistic aspects of the chemoselective catalyzed reaction of [Cp*Rh(bpy)H]+ with the biomimetic NAD+ analogues, N-benzylnicotinamide triflate, 1, and β-nicotinamide ribose-5′-methyl phosphate, 2, in the conversion to their 1,4-NADH analogues, 1,4-dihydro-N-benzylnicotinamide, 4, and β-1,4-dihydronicotinamide-5′-ribose methyl phosphate, 5. This reaction was in tandem with the 1,4-NADH dependent HLADH-Zn(II)-catalyzed reduction of achiral ketones to chiral S-alcohols. The [Cp*Rh(bpy)H]+ complex, and not its equilibrium tautomer, [η4-Cp*HRh(bpy)]+, was found to control the hydride transfer during the biomimetic NAD+/1,4-NADH conversion, through the non-covalent interactions of the biomimetic co-factors with [Cp*Rh(bpy)H]+. The thermodynamics and kinetics for the chiral reduction of the Zn(II) bound ketones, 2-pentanone and 4-phenyl-2-butanone, with co-factor, 4, catalyzed by Zn(SCH3)2(Imidazole), a core model of the Zn(II)-based catalytic center of HLADH, was also investigated by the evaluation of two possible reaction pathways: (1) formation of a ZnH from the C4-H hydride transfer of co-factor, 4, followed by reaction of the postulated ZnH with the bound 2-pentanone or 4-phenyl-2-butanone substrate, and (2), the direct C4-H transfer to the bound achiral ketone substrates, to provide the dominant chiral alcohols, S-2-pentanol or S-4-phenyl-2-butanol. The latter pathway was found most viable, and DFT calculations also revealed an essential η2-coordination of the 5,6 double bond of co-factor, 4, to the HLADH-Zn(II) metal ion center, upon imidazole decomplexation, providing an asymmetric differentiation of S-η2-5,6-1,4-NADH-Zn(II) binding. A proposed new paradigm for the Zn(II)'s non-innocent role in the HLADH-Zn(II) biocatalysis reduction mechanism, for enantioselective hydride transfer to a Zn(II) bound ketone, providing S-alcohols.

Engineering the biomimetic cofactors of NMNH for cytochrome P450 BM3 based on binding conformation refinement.

Cytochrome P450 BM3 (BM3) is an important oxidoreductase that is widely used in drug synthesis, chemical synthesis, and other industries. However, as BM3 unquestionably increases costs by consuming a natural cofactor that unstably provides electrons, an alternative biomimetic cofactor with simpler structures represented by nicotinamide mononucleotide (NMNH) has been utilized. Currently, few reports exist on artificially modified BM3 enzymes using NMNH, especially regarding theoretical simulation and calculation. With the cognition of the mechanism in mind, we propose a strategy that optimizes and refines catalytic conformation. Based on constrained molecular dynamics simulation, the distance between N-5 of FAD flavin and C-4 of NMNH is used as a cue for the determination of improved conformation, and the potential positive mutants are subsequently screened virtually in accordance with binding free energy requirements. As a result, the Kcat/KM values of the favorable mutant S848R increased to 205.38% compared to the wild-type BM3 with NMNH. These data indicate that our strategy can be applied for the specific utilization of biomimetic cofactors by oxidoreductases represented by BM3.

Rational design of electroactive redox enzyme nanocapsules for high-performance biosensors and enzymatic biofuel cell

The potential application of biodevices based on enzymatic bioelectrocatalysis are limited by poor stability and electrochemical performance. To solve the limitation, modifying enzyme with functional polymer to tailor enzyme function is highly desirable. Herein, glucose oxidase (GOx) was chosen as a model enzyme, and according to the chemical structure of GOx cofactor (flavin adenine dinucleotide, FAD), we customize a biomimetic cofactor containing vinyl group (SFAD) for GOx, and prepared an GOx nanocapsule via in-situ polymerization. The characterization of particle size distribution, TEM, fluorescence and electrochemical performance indicated the successful formation of electroactive GOx nanocapsule with SFAD-containing polymeric network (n (GOx-SFAD-PAM)). The network can act as an electronic “highway” to link the active site with electrode, with capability to accelerate electron transfer as well as enhanced GOx stability. Further investigation of bioelectrocatalysis shows that n (GOx-SFAD-PAM)-based biosensor has low detection potential (−0.4 vs. Ag/AgCl), high sensitivity (64.97 μAmM−1cm−2), good anti-interference performance, quick response (3⁓5s) and excellent stability, and that n (GOx-SFAD-PAM)-based enzymatic biofuel cell (EBFC) has the high maximum power density (1011.21 μWcm−2), which is a 385-fold increase over that of native GOx-based EBFC (2.62 μWcm−2). This study suggests that novel enzyme nanocapsule with electroactive polymeric shell might provide a prospective solution for the performance improvement of enzymatic bioelectrocatalysis-based biodevices.

Platinum nanoparticle-deposited multi-walled carbon nanotubes as a NADH oxidase mimic: characterization and applications.

The effective regeneration of bioactive NAD+ plays an important role in numerous dehydrogenase-dependent applications including biocatalysis and biosensing. However, this process usually suffers from high thermodynamic barrier, instability and high cost associated with natural enzymes. The emergence of nanomaterials with enzyme mimic characteristics has offered a potential alternative to many enzyme-catalyzed processes. Platinum nanoparticles (PtNPs), for example, have been extensively studied for their peroxidase- and oxidase-like activities. However, their behavior as a NADH oxidase mimic has barely been characterized in detail. Herein, we report a facile approach for preparing PtNP-deposited multi-walled carbon nanotubes (PtNPs@MWCNTs) as the nanozyme for NADH oxidation. Its enzymatic activity was investigated in depth, revealing that it is a NADH oxidase instead of a peroxidase and the catalytic process generates O2˙-, rather than OH˙ or 1O2, from dissolved O2. The recovery yield of bioactive NAD+ regeneration by the nanozyme could reach ∼100% with a total turnover number of ∼6000. Besides, it exhibited terrific electrochemical performance for NADH oxidation and sensing by greatly boosting the response and lowering the oxidation overpotential. It could also work on biomimetic cofactors with even higher activity. Finally, xylose dehydrogenase was immobilized with the nanozyme to constitute a hybrid bioelectrode for xylose sensing. The biosensor had a xylose detecting range of 5-400 μM with the limit of detection as low as 1 μM and can retain its performance after being reused several times. Our results suggest that the PtNPs@MWCNTs characterized as a NADH oxidase nanozyme hold great promise in the applications of biocatalysis and biosensing, which intensively deal with dehydrogenases and natural or biomimetic cofactors.

1,4-NADH Biomimetic Co-Factors with Horse Liver Alcohol Dehydrogenase (HLADH), Utilizing [Cp*Rh(bpy)H](OTf) for Co-factor Regeneration, Do in Fact, Produce Chiral Alcohols from Reactions with Achiral Ketones

In this Catalysts Comment Article, we will present our latest published results [...]

Synthetic Biomimetic Coenzymes and Alcohol Dehydrogenases for Asymmetric Catalysis

Redox reactions catalyzed by highly selective nicotinamide-dependent oxidoreductases are rising to prominence in industry. The cost of nicotinamide adenine dinucleotide coenzymes has led to the use of well-established elaborate regeneration systems and more recently alternative synthetic biomimetic cofactors. These biomimetics are highly attractive to use with ketoreductases for asymmetric catalysis. In this work, we show that the commonly studied cofactor analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) can be used with alcohol dehydrogenases (ADHs) under certain conditions. First, we carried out the rhodium-catalyzed recycling of BNAH with horse liver ADH (HLADH), observing enantioenriched product only with unpurified enzyme. Then, a series of cell-free extracts and purified ketoreductases were screened with BNAH. The use of unpurified enzyme led to product formation, whereas upon dialysis or further purification no product was observed. Several other biomimetics were screened with various ADHs and showed no or very low activity, but also no inhibition. BNAH as a hydride source was shown to directly reduce nicotinamide adenine dinucleotide (NAD) to NADH. A formate dehydrogenase could also mediate the reduction of NAD from BNAH. BNAH was established to show no or very low activity with ADHs and could be used as a hydride donor to recycle NADH.

Characterization of Biomimetic Cofactors According to Stability, Redox Potentials, and Enzymatic Conversion by NADH Oxidase from Lactobacillus pentosus

Oxidoreductases are attractive biocatalysts that convert achiral substrates into products of higher value, but they are also for the most part dependent on nicotinamide cofactors. Recently, biomimetic nicotinamide derivatives have received attention as less costly alternatives to natural cofactors. However, recycling of biomimetics is still challenging because there are only limited opportunities. Here, we have characterized various biomimetic cofactors with regard to stability and redox potentials to find the best alternative to natural cofactors. Further, the cofactor spectrum of NADH oxidase from Lactobacillus pentosus (LpNox) could be expanded, and the enzymatic activity was also compared to activities with different small-molecule catalysts. As a result, we succeeded in identifying several strategies for regeneration of oxidized biomimetics.

Enzymatic Reduction of Nicotinamide Biomimetic Cofactors Using an Engineered Glucose Dehydrogenase: Providing a Regeneration System for Artificial Cofactors

The increasing demand for chiral compounds supports the development of enzymatic processes. Dehydrogenases are often the enzymes of choice due to their high enantioselectivity combined with broad substrate acceptance. However, their requirement on costly NAD(P)/H as cofactor has sparked interest in the development of biomimetic derivatives that are easy to synthesize and, therefore, less expensive. Until now, few reactions with biomimetics have been described and regeneration is limited to nonenzymatic means, which are not suitable for incorporation and in situ approaches. Herein, we describe a regeneration enzyme, glucose dehydrogenase from Sulfolobus solfataricus (SsGDH), and demonstrate its activity with different biomimetics with the structure nicotinamide ring-alkyl chain-phenyl ring. Subsequent enzyme engineering resulted in the double mutant SsGDH Ile192Thr/Val306Ile, which had a 10-fold higher activity with one of the biomimetics compared with the wild-type enzyme. Using this engineered variant in...

Bioorganometallic chemistry: Co-factor regeneration, enzyme recognition of biomimetic 1,4-NADH analogs, and organic synthesis; tandem catalyzed regioselective formation of N-substituted-1,4-dihydronicotinamide derivatives with [Cp*Rh(bpy)H]+, coupled to chiral S-alcohol formation with HLADH, and engineered cytochrome P450s, for selective C-H oxidation reactions

Two novel tandem catalysis approaches for the chiral synthesis of S-alcohols from reduction of their prochiral ketones with Horse Liver Alcohol Dehydrogenase (HLADH), and selective C-H oxidation reactions with protein engineered Cytochrome P450s, are presented. We utilized a co-factor regeneration procedure with three biomimetic NAD+ models that do not contain the pyrophosphate, nor the adenosine group, and either/or a ribose, N-1-benzylnicotinamide triflate, 1, N-4-methoxybenzylnicotinamide triflate, 2, and β-nicotinamide-5′-ribose methyl phosphate, 3, in conjunction with in situ formed [Cp*Rh(bpy)H]+ from [Cp*Rh(bpy)(H2O)]2+ (Cp* = η5-C5Me5, bpy = 2,2'-bipyridyl) and the hydride source, sodium formate, to regioselectively provide their 1,4-NADH analogs, N-benzyl-1,4-dihydronicotinamide, 4, N-4-methoxybenzyl-1,4-dihydronicotinamide, 5, and 1,4-dihydronicotinamide-5′-ribose methylphosphate, 6. Surprisingly, the 1,4-NADH biomimics, 4 and 6, were recognized, in the second tandem catalysis approach, by the natural 1,4-NADH dependent enzyme, HLADH, for catalyzed, highly enantioselective conversions of prochiral ketones to chiral S-alcohols. For example, with phenethylmethyl ketone and benzylmethyl ketone, the corresponding chiral alcohols were formed in >93% ee (S-enantiomer). Thus, 1,4-NADH biomimetic model recognition by HLADH does not significantly depend on the presence of the ribose, pyrophosphate, or adenosine groups to provide chiral products. We will also propose a plausible active site (HLADH)Zn-H intermediate, generated via a hydride transfer from bound 4/6 to Zn, for the enzymatic reduction of prochiral aryl/alkyl ketones to their chiral aryl/alkyl S-alcohols. Furthermore, the use of protein engineered cytochrome P450 enzymes provided improved molecular recognition of the above mentioned 1,4-NADH biomimetic co-factors, 4 and 5, for selective C-H oxidation reactions. For example, 1,4-NADH dependent mutants of natural 1,4-NAD(P)H dependent P450 BM-3 and 1,4-NADH dependent P450 CAM, with biomimetic co-factors 4 and 5, provided selective oxidation of p-nitrophenoxydecanoic acid to ω-oxydecanocarboxylic acid and p-nitrophenol, via C-H hydroxylation and β-hydrogen elimination, while oxidation of camphor provided hydroxycamphor, respectively. We will discuss the various parameters that effect molecular recognition of the biomimics, including protein engineering of both P450 BM-3 and P450 CAM enzymes, while determining the effect of the co-factor regeneration procedure on HLADH and P450 enzyme activity. These important observations have created new paradigms for the synthesis of organic compounds of interest, with the economically more favorable biomimics of NAD+, 1,4-NADH, and 1,4-NAD(P)H as co-factors, in tandem with the use of [Cp*Rh(bpy)(H)]+ as a regioselective catalytic reagent for co-factor regeneration.

A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors.

The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.

Mass effect of redox reactions: A novel mode for surface plasmon resonance-based bioanalysis.

The pursuit of more specific and sensitive response is a perpetual goal for modern bioassays. This work proposed a novel label-free strategy about redox-related mass effect based on the surface plasmon resonance (SPR) technique for ultrasensitive determination of DNA. The protocol starts with the modification of SPR gilded disk with the capture DNA (cDNA). After the conjugation of immobilized cDNA with the target DNA (tDNA), the hybridization chain reaction was triggered by the introduction of mutual partial complementary primers to elongate the terminal into a nanoscale duplex. As it is reported that porphyrin could intercalate into the grooves of the double-stranded DNA (dsDNA) scaffold, multiple positive-charged Fe(III)meso-tetra(N-methyl-4-pyridyl) porphine (FeTMPyP) with symmetric structure were uptaken for in situ formation of porphyrin-dsDNA complex. Given FeTMPyP a highly efficient catalysis for the peroxide reduction, its presence as a biomimetic cofactor was validated via circular dichroism and UV-vis spectroscopy, demonstrating a tight binding as well as high catalytic activity and stability. Using 4-chloro-1-naphthol as a proton donor, the catalytic reduction of H2O2 would oxidize it into insoluble benzo-4-chloro-hexadienone, which simultaneously deposited on the heterogeneous interface, leading to a significant amplification in both SPR response and topological height profile. The signal increment was proportional to the concentration of tDNA, thus an ultrasensitive SPR-based DNA assay was developed with a linear range over four orders of magnitudes and a sub-femtomolar detection limit of 0.73 fM. The developed methodology exemplifies a different way of thinking about mass-sensing modes, extending conventional SPR-based DNA analysis to relevant biomedical applications.

Enzymatic biofuel cells utilizing a biomimetic cofactor

The performance of immobilized enzyme systems is often limited by cofactor diffusion and regeneration. Here, we demonstrate an engineered enzyme capable of utilizing the minimal cofactor nicotinamide mononucleotide (NMN(+)) to address these limitations. Significant gains in performance are observed with NMN(+) in immobilized systems, despite a decreased turnover rate with the minimal cofactor.

Bioorganometallic chemistry: biocatalytic oxidation reactions with biomimetic NAD +/NADH co-factors and [Cp*Rh(bpy)H] + for selective organic synthesis

The biocatalytic, regioselective hydroxylation of 2-hydroxybiphenyl to the corresponding catechol was accomplished utilizing the monooxygenase 2-hydroxybiphenyl 3-monooxygenase (HbpA). The necessary natural 1,4-dihydronicotinamde adenine dinucleotide (NADH) co-factor for this biocatalytic process was replaced by a biomimetic co-factor, N-benzyl-1,4-dihydronicotinamide, 1b. The interaction between the flavin (FAD) containing HbpA enzyme and the corresponding biomimetic NADH compound, N-benzyl-1,4-dihdronicotinamide, 1b, for hydride transfer, was shown to readily occur. The in situ recycling of the reduced NADH biomimic 1b from 1a was accomplished with [Cp*Rh(bpy)H](Cl); however, productive coupling of this regeneration reaction to the enzymatic hydroxylation reaction was not totally successful, due to a deactivation process concerning the HbpA enzyme peripheral groups; i.e., –SH or –NH 2 possibly reacting with the precatalyst, [Cp*Rh(bpy)(H 2O)](Cl) 2, and thus inhibiting the co-factor regeneration process. The deactivation mechanism was studied, and a promising strategy of derivatizing these peripheral –SH or –NH 2 groups with a polymer containing epoxide was successful in circumventing the undesired interaction between HbpA and the precatalyst. This latter strategy allowed tandem co-factor regeneration using 1a or 2a, [Cp*Rh(bpy)(H 2O)](Cl) 2, and formate ion, in conjunction with the polymer bound, FAD containing HbpA enzyme to provide the catechol product.