Nicotinamide-dependent Enzymes Research Articles

Flavoenzymes for biocatalysis.

Flavoenzymes are broadly employed as biocatalysts for a large variety of reactions, owing to the chemical versatility of the flavin cofactor. Oxidases set aside, many flavoenzymes require a source of electrons in form of the biological reductant nicotinamide NAD(P)H in order to initiate catalysis via the reduced flavin. Chemists can take advantage of the reactivity of reduced flavins with oxygen to carry out monooxygenation reactions, while the reduced flavin can also be used for formal hydrogenation reactions. The main advantage of these reactions compared to chemical approaches is the frequent regio-, chemo- and stereo-selectivity of the biocatalysts, which allows the synthesis of chiral molecules in optically active form. This chapter provides an overview of the variety of biocatalytic processes that have been developed with flavoenzymes, with a particular focus on nicotinamide-dependent enzymes. The diversity of molecules obtained is highlighted and in several cases, strategies that allow control of the stereochemical outcome of the reactions are reviewed.

Broadening the repertoire of microbial aldo-keto reductases: cloning and characterization of AKR3B4 from Rhodotorula mucilaginosa LSL strain

Aldo-keto reductases (AKRs) are nicotinamide-dependent enzymes that catalyze the transformation of aldehydes and ketones into alcohols. They are spread across all phyla, and those from microbial origin have proved to be highly robust and versatile biocatalysts. In this work, we have discovered and characterized a microbial AKR from the yeast Rhodotorula mucilaginosa by combining genome-mining and expression assays. The new enzyme, named AKR3B4, was expressed by a simple protocol in very good amounts. It displays a selective substrate profile exclusively transforming aldehydes into alcohols. Also, AKR3B4 shows very good stability at medium temperatures, in a broad range of pH values and in the presence of green organic solvents. Conversion assays demonstrate it is an excellent biocatalyst to be used in the synthesis of aromatic alcohols, and also to produce furan-3-ylmethanol and the valuable sweetener xylitol. These results show that AKR3B4 displays attractive features so as to be used in chemoenzymatic processes.

Catalytic promiscuity enabled by photoredox catalysis in nicotinamide-dependent oxidoreductases

Strategies that provide enzymes with the ability to catalyse non-natural reactions are of considerable synthetic value. Photoredox catalysis has proved adept at expanding the synthetic repertoire of existing catalytic platforms, yet, in the realm of biocatalysis it has primarily been used for cofactor regeneration. Here we show that photoredox catalysts can be used to enable new catalytic function in nicotinamide-dependent enzymes. Under visible-light irradiation, xanthene-based photocatalysts enable a double-bond reductase to catalyse an enantioselective deacetoxylation. Mechanistic experiments support the intermediacy of an α-acyl radical, formed after the elimination of acetate. Isotopic labelling experiments support nicotinamide as the source of the hydrogen atom. Preliminary calculations and mechanistic experiments suggest that binding to the protein attenuates the reduction potential of the starting material, an important feature for localizing radical formation to the enzyme active site. The generality of this approach is highlighted with the radical dehalogenation of α-bromoamides catalysed by ketoreductases with Eosin Y as a photocatalyst.

Recent advances in imine reductase-catalyzed reactions.

Imine reductases are nicotinamide-dependent enzymes that catalyze the asymmetric reduction of various imines to the corresponding amine products. Owing to the increasing roles of chiral amines and heterocyclic compounds as intermediates for pharmaceuticals, the demand for novel selective synthesis strategies is vitally important. Recent studies have demonstrated the discovery and structural characterization of a number of stereoselective imine reductase enzymes. Here, we highlight recent progress in applying imine reductases for the formation of chiral amines and heterocycles. It particularly focuses on the utilization of imine reductases in reductive aminations of aldehydes and ketones with various amine nucleophiles, one of the most powerful reactions in the synthesis of chiral amines. Second, we report on the synthesis of saturated substituted N-heterocycles by combining them with further biocatalysts, such as carboxylic acid reductases, oxidases or transaminases. Finally, we summarize the latest applications of imine reductases in the promiscuous asymmetric hydrogenation of a highly reactive carbonyl compound and the engineering of the cofactor specificity from NADPH to NADH.

Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light.

Enzymes are ideal for use in asymmetric catalysis by the chemical industry, because their chemical compositions can be tailored to a specific substrate and selectivity pattern while providing efficiencies and selectivities that surpass those of classical synthetic methods. However, enzymes are limited to reactions that are found in nature and, as such, facilitate fewer types of transformation than do other forms of catalysis. Thus, a longstanding challenge in the field of biologically mediated catalysis has been to develop enzymes with new catalytic functions. Here we describe a method for achieving catalytic promiscuity that uses the photoexcited state of nicotinamide co-factors (molecules that assist enzyme-mediated catalysis). Under irradiation with visible light, the nicotinamide-dependent enzyme known as ketoreductase can be transformed from a carbonyl reductase into an initiator of radical species and a chiral source of hydrogen atoms. We demonstrate this new reactivity through a highly enantioselective radical dehalogenation of lactones-a challenging transformation for small-molecule catalysts. Mechanistic experiments support the theory that a radical species acts as an intermediate in this reaction, with NADH and NADPH (the reduced forms of nicotinamide adenine nucleotide and nicotinamide adenine dinucleotide phosphate, respectively) serving as both a photoreductant and the source of hydrogen atoms. To our knowledge, this method represents the first example of photo-induced enzyme promiscuity, and highlights the potential for accessing new reactivity from existing enzymes simply by using the excited states of common biological co-factors. This represents a departure from existing light-driven biocatalytic techniques, which are typically explored in the context of co-factor regeneration.

Characterization of azido-NAD + to assess its potential as a two-dimensional infrared probe of enzyme dynamics

Enzyme active-site dynamics at femtosecond to picosecond time scales are of great biochemical importance, but remain relatively unexplored due to the lack of appropriate analytical methods. Two-dimensional infrared (2D IR) spectroscopy is one of the few methods that can examine chemical biological motions at this time scale, but all the IR probes used so far were specific to a few unique enzymes. The lack of IR probes of broader specificity is a major limitation to further 2D IR studies of enzyme dynamics. Here we describe the synthesis of a general IR probe for nicotinamide-dependent enzymes. This azido analog of the ubiquitous cofactor nicotinamide adenine dinucleotide is found to be stable and bind to several dehydrogenases with dissociation constants similar to that for the native cofactor. The infrared absorption spectra of this probe bound to several enzymes indicate that it has significant potential as a 2D IR probe to investigate femtosecond dynamics of nicotinamide-dependent enzymes.

Transient oxidation as a mechanistic strategy in enzymatic catalysis

Enzymes that employ a transient oxidation mechanism catalyze transformations that are overall redox neutral, but involve intermediates that have a higher oxidation state than the substrates or products. An oxidation/reduction sequence may be used directly to promote isomerization reactions or indirectly to permit the formation of stabilized intermediates such as carbanions. This review will focus on three recent examples of nicotinamide-dependent enzymes that have been found to employ transient oxidation during catalysis: ADP-L-glycero-D-manno-heptose 6-epimerase, GDP-mannose 3,5-epimerase, and the 6-phosphoglucosidases from family 4. These enzymes are remarkable in their ability to catalyze either nonstereospecific hydride transfers or multiple chemical steps within a single active site.

Synthesis and utility of 14C-labeled nicotinamide cofactors

A new method for the synthesis of the reduced form of β-nicotinamide [U- 14C]adenine dinucleotide 2 ′-phosphate([Ad- 14C]NADPH) is presented. The present synthesis results in a radioactive material with a specific activity that is greater than 220 mCi/mmol. This method could easily be adapted for syntheses of 14C-labeled NADH, NADP +, or any nicotinamide cofactors with radiolabels in other positions. Since these cofactors are so ubiquitous, the use and applications of such labeled material has broad implications. The utility of the labeled cofactor for determination of substrates for nicotinamide-dependent enzymes in the nano- to femtomole scale, in alternative enzymatic assays, and in kinetic isotope effect studies is discussed.

Synthesis of R and S tritiated reduced β-nicotinamide adenine dinucleotide 2 ′ phosphate

Nicotinamides are ubiquitous cofactors used by many biological systems as redox agents. Stereospecifically labeled cofactors are useful in many studies of nicotinamide-dependent enzymes. Enzyme-directed synthesis of these cofactors is rather common but their stability imposes significant challenges on yield, purity, and preservation. This paper presents the stereospecific synthesis of reduced R- and S-[4- 3H] β-nicotinamide adenine dinucleotide 2 ′ phosphate (NADPH). The method of Valera et al. [Biochem. Biophys. Res. Commun. 148 (1987) 515] was modified to a synthetic procedure that produces both isotopic diastereomers within 2 h with an improved yield of 75–90% after purification and lyophilization. In the synthesis, [4- 3H]NADP + was generated as an intermediate (which can be isolated if desired). The specific radioactivities reported here are 2.7 and 1.1 Ci/mmol for the S and R diastereomers, respectively. Specific radioactivities ranging from carrier-free to trace labeling can be achieved with a minor change to the procedure.

Purification, analysis, and preservation of reduced nicotinamide adenine dinucleotide 2 ′-phosphate

Nicotinamide-containing cofactors are ubiquitous in biological systems. Consequently, numerous assays have been developed to study such systems that involve a variety of derivatives and isotopically labeled forms of these cofactors. Often the nicotinamide ring is labeled at the C-4 position which is directly involved in the hydride transfer chemistry catalyzed by nicotinamide-dependent enzymes. A label remote from the reactive center is often also required to follow the course of a reaction or the location of the cofactor. Since the necessary labeling pattern can be as unique as the designed experiment, these cofactors need to be synthesized, analyzed, and, most preferably, preserved. The micro-scale preservation of reduced nicotinamides has long been a challenge due to the inherent lability of the reduced cofactors. Furthermore, it has been found that the reduced 2 ′-phosphorylated cofactor is even less stable (i.e., reduced nicotinamide adenine dinucleotide phosphate (NADPH) is more labile than reduced nicotinamide adenine dinucleotide). Presented here are methods that were established to purify nicotinamide cofactors via reverse-phase high-performance liquid chromatography (HPLC) and, most importantly, to stabilize NADPH under optimal conditions for long-term storage. Additionally, an analytical HPLC method which achieves 7-min resolution between oxidized and reduced cofactors was developed. This method also results in over 4-min resolution of these nicotinamide cofactors from various derivatives of folic acid. This analysis affords a new analytical assay for the dihydrofolate reductase-catalyzed reaction and several dehydrogenases involved in folic acid metabolism.

The oxidation of cyclopropyl benzene by rat liver microsomal cytochrome P-450: an unusual triple oxidation of a substrate

The mechanism of hydroxylation of both aliphatic and aromatic molecules by cytochromes P450 and related chemical model systems has been studied in depth recently [l] and a wide range of approaches has been used. For example, Groves [2] has demonstrated the radical character of some model hydroxylating systems based upon tetraphenyl porphyrin by observing the ring opening of the cyclopropane ring in bicyclo(4,2,0)heptane and we have used a related approach to investigate the possibility of radical intermediates in hydrogen transfer reactions mediated by nicotinamide-dependent enzymes [3]. It was therefore of interest to attempt to extend the applicability of the cyclopropyl group as a probe for radical intermediates in aromatic substitution by examining the hydroxylation of cyclopropylbenzene by a cytochrome P-450 and related chemical systems. In this paper, we describe the reactions of cyclopropylbenzene with rat liver microsomal cytochrome P-450: related chemical reactions are discussed elsewhere [4].

The influence of nicotinamide on the urinary excretion of tryptophan metabolites in Hodgkin's disease.

Abstract The urinary excretion of tryptophan metabolites was studied in 10 patients with Hodgkin's disease. As previously found, kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid were spontaneously excreted in abnormally high amounts, appearing to be a constant characteristic in patients with Hodgkin's disease. Tryptophan loading produced an abnormal excretion of 10 metabolites in all, and normal excretion was only partially restored by treatment with nicotinamide administration before a second tryptophan test in the same patients. The effect of nicotinamide on the excretion pattern appeared to differ qualitatively and quantitatively from that obtained using vitamin B6 administration. These data show the existence, in Hodgkin's disease, of a deficiency of nicotinamide-dependent enzymes acting on the tryptophan → nicotinic acid pathway, besides that correlated to vitamin B6.