A new approach for using cofactor dependent enzymes: example of alcohol dehydrogenase

Sicsic, Sames; Durand, Philippe; Langrené, Sylvie; Goffic, François Le

doi:10.1016/0014-5793(84)81188-6

Cited by 19 publications

(9 citation statements)

References 6 publications

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“…The present biomimetics all lack the adenine dinucleotide moiety that is part of the natural cofactor NADH. Sicsic et al discovered that the addition of adenosine or adenosine-5′ phosphate to enzymatic reactions increases the activity of horse liver alcohol dehydrogenase with nicotinamide mononucleotide or nicotinamide mononucleoside . In our experiments, the addition of 5 mM adenosine or adenosine-5′ phosphate to the reaction of Ss GDH with BNA + , P2NA + , or P3NA + and d -glucose did not increase specific activity.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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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 combination with an enoate reductase from Thermus scotoductus resulted in the first enzyme-coupled regeneration process for biomimetic cofactor without ribonucleotide or ribonucleotide analogue and full conversion of 10 mM 2-methylbut-2-enal with 1-phenethyl-1,4-dihydropyridine-3-carboxamide as cofactor.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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“…These examples are important steps towards the use of NCBs with dehydrogenases, and more specifically ADHs, which would be advantageous for large scale applications, although NCB recycling and stability is still in its early stages [36]. In a previous study, cofactors NR, NMN, and carbocyclic analogues showed no observable activity in the HLADH-catalyzed oxidation of ethanol [37,38], corroborated by computational studies on the mechanism for hydride transfer in HLADH [39].…”

Section: Introductionmentioning

confidence: 77%

Synthetic Biomimetic Coenzymes and Alcohol Dehydrogenases for Asymmetric Catalysis

Josa-Culleré¹,

Lahdenperä²,

Ribaucourt³

et al. 2019

Catalysts

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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.Catalysts 2019, 9, 207 2 of 11 or aldehydes to the corresponding (enantioenriched) alcohols through the use of one equivalent of nicotinamide adenine dinucleotide cofactor NAD(P). ADHs are known to be specific to either the phosphorylated NADP or non-phosphorylated NAD, although a few have been found to accept both [1-3]. These cofactors act as hydride acceptor and donor intermediates between the substrate and product.Enzyme recognition of NAD(P), usually by a cofactor (binding) motif such as the Rossmann fold, is important for cellular processes, however when using in vitro systems, the adenine dinucleotide moiety of the cofactor becomes obsolete and disadvantageous, being prone to hydrolysis [4]. Synthetic nicotinamide coenzyme biomimetics (NCBs) have been shown to replace NAD(P)H in flavin-dependent enzymes such as nitroreductase and NAD(P)H quinone oxidoreductase [5-7], a cytochrome P450 BM3 variant [8], ene-reductases [9][10][11][12][13][14][15][16], and styrene monooxygenase [17]. In all cases described, a flavin was involved as an electron mediator [18][19][20][21][22].Jones pioneered the use of synthetic cofactor analogues with horse liver ADH (HLADH) [23][24][25][26], followed by Fish [27] and others [28,29]. A dehydrogenase from the aldo-reductase superfamily from Pyrococcus furiosus (AdhD) was engineered to increase catalytic efficiency towards nicotinamide mononucleotide (NMN, Figure 1A) [30,31]. Acyclic analogues of NAD ( Figure 1B) were shown not only to be used with HLADH, but also to alter the substrate specificity of the enzyme [32]. More recently, the group of Sieber showed the use of synthetic NCBs with an NADH oxidase [33,34], and a glucose dehydrogenase...

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“…However, whether this redox cycle is well insulated from the numerous endogenous ADHs in E. coli crude lysates has not been tested. On the basis of previous studies, XenA can accept reducing power in the forms of both NADP(H) and reduced NMN + (NMNH), while endogenous ADHs greatly prefer NAD(P)H over NMNH, likely because of NMNH’s truncated structure which lacks the adenosine moiety “handle” of NAD(P)H. ,− Thus, we hypothesized that when using GDH wild type (WT) to derive reducing power from glucose, both XenA and ADHs will receive electrons because NAD(P) + are the cycling cofactor (Figure B). On the other hand, when GDH Ortho is used with the supplementation of NMN + , only XenA will receive electrons because the stringent cofactor specificity of GDH Ortho allows it to only generate NMNH but not NAD(P)H (Figure C).…”

Section: Resultsmentioning

confidence: 99%

Aldehyde Production in Crude Lysate- and Whole Cell-Based Biotransformation Using a Noncanonical Redox Cofactor System

Richardson

Black

2020

ACS Catal.

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It is challenging to biosynthesize industrially important aldehydes, which are readily consumed by the numerous alcohol dehydrogenases (ADHs) in cells. In this work, we demonstrate that a nicotinamide mononucleotide (NMN + )-dependent redox cofactor cycling system enables aldehyde accumulation in Escherichia coli crude lysates and whole cells. By specifically delivering reducing power to a recombinant enoate reductase, but not to endogenous ADHs, we convert citral to citronellal with minimal byproduct formation (97−100% and 83% product purity in crude lysate-and whole cell-based biotransformation, respectively). We envision the system's universal application to lowering the noise in biomanufacturing by silencing the host's metabolic background.

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A new approach for using cofactor dependent enzymes: example of alcohol dehydrogenase

Cited by 19 publications

References 6 publications

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

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

Synthetic Biomimetic Coenzymes and Alcohol Dehydrogenases for Asymmetric Catalysis

Aldehyde Production in Crude Lysate- and Whole Cell-Based Biotransformation Using a Noncanonical Redox Cofactor System

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