BBA - Proteins and Proteomics 1866 (2018) 327–347

Contents lists available at ScienceDirect

BBA - Proteins and Proteomics

journal homepage: www.elsevier.com/locate/bbapap

Review Review of NAD(P)H-dependent : Properties, engineering T and application ⁎ Lara Sellés Vidal, Ciarán L. Kelly, Paweł M. Mordaka, John T. Heap

Centre for Synthetic Biology and Innovation, Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom

ARTICLE INFO ABSTRACT

Keywords: NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a coupled to the oxidation NAD(P)H-dependent oxidoreductases or reduction, respectively, of a nicotinamide adenine dinucleotide NAD(P)H or NAD(P)+. NAD(P)H- Metabolic engineering dependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic Protein engineering pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze reac- Directed evolution tions, they have been used as key components in a wide range of applications, including substrate utilization, the promiscuity synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-depen- Substrate specificity dent oxidoreductases to make them more suitable for particular applications. Here, we review the main prop- erties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their mod- ification and application.

1. Introduction electron transport capability as it is located far from the electron transfer region (Fig. 2). However, the phosphate group modifies the Oxidoreductases (Enzyme Commission [EC] primary class 1) cata- structure of the cofactor, which allows different to have dif- lyze the oxidation of one chemical species (a reducing agent or electron ferent specificities for NADH/NAD+ and NADPH/NADP+, thereby al- donor) with the concurrent reduction of another (an oxidizing agent or lowing these to act as two equivalent but independent redox systems. − − electron acceptor) in the form A +B→ A+B and comprise almost This has a physiological function, allowing different a redox poise one third of all enzymatic activities registered in the BRaunschweig (degree of reduction of the cofactor pool) to be maintained in the two ENzyme Database, BRENDA (Fig. 1). Oxidoreductases can act on a wide systems, and independent fluxes. Typically, at least in heterotrophs, range of both organic substrates including alcohols, amines and ketones enzymes of catabolic pathways use NADH/NAD+, while NADPH/ and inorganic substrates including small anions such as sulfite, and NADP+ is the preferred cofactor for anabolism [3,4]. The redox poise of metals such as mercury. the NADH/NAD+ pool depends upon the availability and redox state of NAD(P)H-dependent oxidoreductases are able to oxidize a substrate external electron acceptors and of substrates. This variation in redox − by transferring a hydride (H ) group to a nicotinamide adenine dinu- poise can be observed for example in Escherichia coli growing via cleotide cofactor (either NAD+ or NADP+), resulting in the reduced aerobic respiration, anaerobic respiration, or anaerobic fermentation, form NADH or NADPH (Fig. 2), and make up over 50% of all oxidor- where factors such as the oxygen availability when growing aerobically, eductase activities registered in the BRENDA (Fig. 1). There are over or the redox potential of other electron acceptors used when growing 150,000 different sequences annotated as or predicted to be NAD(P)H- anaerobically, affect the steady-state NADH/NAD+ ratio [5]. In con- dependent oxidoreductases [1]. trast, the poise of the NADPH/NADP+ pool is maintained in a more NADH/NAD+ and NADPH/NADP+ serve as pools of redox cofactors reduced state in order to more effectively provide reducing power for for the cell. The nicotinamide ring of NADH/NAD+ or NADPH/NADP+ biosynthesis [6]. Accordingly, NADH-dependence is more prevalent is the part of the cofactor directly involved in the transfer of electrons among oxidoreductases acting on smaller molecules, which include during the reactions catalyzed by NAD(P)H-dependent oxidoreductases, most substrates and products of catabolism (Fig. 3). Interestingly, while the C4 carbon atom of the nicotinamide ring acts as the acceptor/ substrates of low molecular weight can be metabolized by a higher donor of a proton [2]. The addition of the phosphate to the 2′-OH group number of enzymatic activities, indicating that smaller substrates have of the adenine ribose ring in NADPH/NADP+ does not modify the a role as central hubs of redox metabolic networks (Fig. 3b). There is

⁎ Corresponding author. E-mail address: [email protected] (J.T. Heap). https://doi.org/10.1016/j.bbapap.2017.11.005 Received 16 August 2017; Received in revised form 27 October 2017; Accepted 8 November 2017 Available online 10 November 2017 1570-9639/ Crown Copyright © 2017 Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

NAD(P)H-dependent oxidoreductases also another, smaller increase of both the absolute number of oxidor- NADPH-dependent oxidoreductases eductase activities (Fig. 3a) and the number of activities per substrate for substrates of molecular weight between 700 and NADH-dependent oxidoreductases 1100 Da, which is partially due to substrates that need to be activated Other oxidoreductases by binding to coenzyme A (CoA) before the corresponding oxidor- eductase can act on them (Fig. 3). Canonically the differing redox states of the two systems (NADH and NADPH) is achieved in heterotrophs using NAD+-dependent glycolysis and the NADP+-dependent pentose phosphate pathway, but there are several variations and alternatives [7], and photoautotrophs generate NADPH using the light-dependent reactions of photosynthesis. The independence of the NADH/NAD+ and NADPH/NADP+ pools also allows different enzymes with different redox cofactors to catalyze key steps of opposite pathways helping to prevent futile cycles [8]. NAD(P)H-dependent oxidoreductases are of great interest from an industrial point of view as they perform the critical steps in the pro- duction of many hard-to-synthesize compounds, under mild conditions. For example, even though several chemical methods have been devel- oped to perform the oxidation of primary alcohols, they are usually laborious and can lead to the formation of toxic products [9], which can be avoided through enzymatic . Additionally, NAD(P)H-de- Fig. 1. Distribution of enzymatic activities registered in BRENDA. Oxidoreductases con- pendent oxidoreductases possess other properties which make them stitute approximately 30% of all the BRENDA enzymatic activities, among which around + + very attractive alternatives to organic chemical synthesis, such as ste- 50% use NADH/NAD and/or NADPH/NADP as a cofactor. The number of enzymatic fi fi activities of each EC class, as well as the number of NADH, NADPH and NAD(P)H-de- reospeci city, regiospeci city and the possibility to tailor them to have pendent oxidoreductases, were obtained from BRENDA by means of manual queries and the appropriate kinetic parameters and the desired substrate specificity plotted with R and the ggplot2 package. [10]. For these reasons, NAD(P)H-dependent oxidoreductases have been used extensively in metabolic engineering for the production of

Fig. 2. Molecular formula of NAD(P)H and NAD(P)+. The NADH molecule contains adenine and nicotinamide nucleosides linked by a pyrophosphate linkage. The nicotinamide ring is the acceptor/donor of the electrons and the C4 (red) of the nicotinamide ring is the acceptor/donor of the proton. NADPH differs from NADH in the substituent at the C2 position of the adenosine moiety, indicated as ‘R’ in the figure, which is a hydroxy in NADH and a phosphate in NADPH. This phosphate group does not alter the ability of the co- factor to transfer electrons. NAD+ is the oxidized form of NADH, and NADP+ is the oxidized form of NADPH.

328 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Fig. 3. a. Number of oxidoreductase activities versus molecular/ atomic weight of the substrates. Substrates are grouped in in- a NAD(P)H-dependent oxidoreductases tervals of 100 Da. Some substrate examples are labelled. A list of NADPH-dependent oxidoreductases enzymes was extracted from the KEGG database using R with NADH-dependent oxidoreductases the KEGGREST package by imposing the condition that either + Other oxidoreductases NAD(P) or NAD(P)H were involved in the reaction as substrate or . Results were plotted with R and the ggplot2 package. b. Number of oxidoreductase activities per substrate versus molecular/atomic weight of the substrates. Data were obtained and plotted in the same way as for Fig. 3a, but the number of oxidoreductase activities in each range of molecular/ atomic weight was divided by the total number of substrates in that interval.

b

low, mid and high value products [11]. a variety of different reactions other than hydrolysis of esters in non- NAD(P)H-dependent oxidoreductases are too numerous and diverse aqueous media, such as transesterification alcoholysis or acidolysis to review exhaustively. Therefore, the reader is provided with a list of [55]. However, more recently other enzymes have been described to recommended papers and reviews relevant to NAD(P)H-dependent have catalytic promiscuity, including NAD(P)H-dependent oxidor- oxidoreductases (Table 1). Here we review this important group of eductases. Certain alcohol dehydrogenases (in particular, alcohol de- enzymes broadly, with an applied perspective. The common functional, hydrogenases from horse liver, Rhodococcus ruber, Ralstonia sp. and structural, mechanistic and physiological properties are described. The Lactobacillus kefir) have been found to catalyze the conversion of oximes various classes of these enzymes are introduced, with emphasis placed (specifically phenylacetaldoxyme) to the corresponding alcohol [56].In on catalytic mechanisms and their structural basis, which can provide another study, an NADPH-dependent ene-reductase (Δ4-3-ketosteroid- important information to guide protein engineering approaches. Ex- 5β-reductase, which reduces a CeC double bond in Δ4-3-ketosteroids) amples of enzymes of applied interest are included, often entailing gained the ability to reduce the carbonyl group of the substrate to a metabolic engineering or protein engineering using common techni- hydroxy group with a single point mutation, an activity that is usually ques. carried out by 3α-hydroxysteroid dehydrogenase [57]. This indicates that the two enzymes probably had a common ancestor from which they diverged to catalyze consecutive steps of the same metabolic 2. Specificity and promiscuity of oxidoreductases pathway. Substrate promiscuity refers to the property of an enzyme of being able to catalyze the same reaction with a range of different but Promiscuity of enzymes can refer to two different concepts: catalytic related substrates [55]. Some are very specific and have just one pos- promiscuity, and specificity. Promiscuous enzymes and enzymes with a sible substrate, such as D-hydroxyisovalerate dehydrogenase from the wide substrate range are of particular interest, since even an initially depsipeptide-producing fungus Fusarium sambucinum [58]. However, low catalytic activity towards a specific substrate can be a good starting many NAD(P)H-dependent oxidoreductases are not strictly specific for point to generate, by means of protein engineering and directed evo- a single substrate, and instead display a varying degree of promiscuity, lution approaches, an efficient catalyst suitable for a specific reaction, being able to act on a set of substrates. The extent to which they are which does not necessarily match the physiological reaction carried out promiscuous differs even between enzymes of the same subclass. For by the original enzyme [54]. example, Thermus sp. ATN1 alcohol dehydrogenase (TADH) exhibits Catalytic promiscuity is the ability of an enzyme to catalyze a activity towards a very large range of different alcohols with multiple second reaction in addition to the main reaction for which it is phy- different functional groups (including both aliphatic and aromatic al- siologically specialized, with a different transition state. , cohols) [59], while Zymomonas mobilis alcohol dehydrogenase 2 is only and in particular lipases, are some of enzymes where catalytic pro- able to oxidize ethanol, 1-propanol and allyl alcohol [60]. miscuity has been most extensively studied, as they are able to catalyze

329 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Table 1 approach. However, it has also become clear that it is not possible to List of recommended papers relevant to NAD(P)H-dependent oxidoreductases. develop a unified approach that can be broadly applied, since the huge variety of substrates upon which this class of enzymes can act as a Subject Reference(s) group is associated with a similarly large variety of structural motifs. In Biotechnological applications [12–14] this section we describe the two main structural features of each class of Alcohol dehydrogenases [15,16] NAD(P)H-dependent oxidoreductases, important conserved catalytic (EC 1.1.1) residues and a brief overview of how these features relate to reaction Aldehyde dehydrogenases [17,18] (EC 1.2.1) mechanisms. Ene-reductases [19] (EC 1.3.1) 3.1. Cofactor binding domain Amino-acid dehydrogenases [20] (EC 1.4.1) One of the key structural motifs of NAD(P)H-dependent oxidor- Oxidoreductases of CHeNH group [21–23] (EC 1.5.1) eductases is the nucleotide binding domain which allows them to re- + Transhydrogenases [24,25] cruit the NAD(P)H/NAD(P) redox cofactor essential for their activity. (EC 1.6.1) The most common fold employed by these enzymes for such purpose is – Oxidoreductases acting on nitrogenous compounds [26 28] the Rossmann fold, but there is a variety of other less common struc- (EC 1.7.1) Oxidoreductases acting on sulfur [29–31] tural motifs which can also bind the redox cofactor, such as the TIM- + (EC 1.8.1) barrel, the dihydroquinoate synthase-like and the FAD/NAD binding Oxidoreductases of diphenols and related substances donors [32] folds [66]. (EC 1.10.1) The Rossmann fold is a common structural motif found in many [33] nucleotide-binding proteins, such as 3-phosphoglycerate dehy- (EC 1.11.1) Oxidoreductases of H2 (Hydrogenases) [34] drogenase and lactate dehydrogenase [67,68]. It is named after Michael (EC 1.12.1) Rossmann, one of the members of the team who first identified the fold Oxygenases and monooxygenases [35–38] in lactate dehydrogenase [69] and later realized it was a conserved (EC 1.13.1 and EC 1.14.1) motif present in other NAD(P)H-binding enzymes [70]. Its most con- – Oxidoreductases of metal ions [39 42] β α (EC 1.16.1) served core consists of two parallel -strands separated by an -helix βαβ fi β Oxidoreductases acting on CH or CH2 groups [43,44] ( motif) (Fig. 4). A tight loop is formed between the rst -strand (EC 1.17.1) and the α-helix, which establishes direct contact with the cofactor. This Oxidoreductases of -sulfur proteins and of flavodoxin [45–49] loop contains the consensus sequence Gly-X-Gly-X-X-Gly/Ala (with X (EC 1.18.1 and EC 1.19.1) being any ) [71,72]. This glycine-rich loop is involved in Oxidoreductases of or compounds [50] (EC 1.20.1) binding to the pyrophosphate of dinucleotides. Computational analysis Reductive dehalogenases [51,52] revealed that a water molecule is invariably present in a very conserved (EC 1.21.1) position bridging the pyrophosphate to the glycine-rich loop [73].The Oxidoreductases reducing C-O-C group [53] cofactor binding domains of NAD(P)H-dependent oxidoreductases ty- (EC 1.23.1) pically contain two Rossmann folds, one interacting with the adenine moiety and the other with the nicotinamide ring. βαβ β There is great variation in the promiscuity of NAD(P)H-dependent The core can be extended in some cases with additional - β β oxidoreductases for the nicotinamide cofactor. Some are highly specific strands to form a larger -sheet with the two core -strands. Usually, all + + β for either NADH/NAD or NADPH/NADP , while others can use both -strands are parallel, although there are some cases where some of the β with varying degrees of preference for one over the other. For example, strands are antiparallel [74]. The segments between the additional - α the previously mentioned D-hydroxyisovalerate dehydrogenase of strands are variable, and can consist of additional -helices, random Fusarium sambucinum, as well as the soluble NADPH-dependent Fe(III) coil regions or complex combinations of short helices and coiled seg- reductase from Geobacter sulfurreducens, are highly specific for NADPH/ ments. NADP+ and are unable to accept NADH/NAD+ [58,61]. At the opposite extreme, the E. coli aldo-keto reductase encoded by the ydjG is 3.2. Catalytic domain highly specific for NADH/NAD+ and is unable to accept NADPH/ NADP. This enzyme is considered to be an exception among the family The other main motif of NAD(P)H-dependent oxidoreductases is the of aldo-keto reductases, which typically either are specific for NADPH/ catalytic domain, which coordinates the substrate and provides the NADP+ or can accept both NADH/NAD+ and NADPH/NADP+ [62]. residues essential for the redox reaction to take place. Due to the very ff Many NAD(P)H-dependent oxidoreductases are described as being able large variety of di erent oxidoreductases and reactions they can cata- + + fi to use both NADH/NAD and NADPH/NADP (Figs. 1 and 3). These lyze, it is impossible to de ne common motifs for all of them (other include the B2FLR2 flavin-containing monooxygenase from Steno- than the frequent presence of other redox cofactors such as FAD, FMN trophomonas maltophilia, which prefers NADH/NAD+, and xylose re- or metallic centres). Thus, here we focus on the catalytic domains of ductase of Neurospora crassa, which prefers NADPH/NADP+ [63,64]. oxidoreductases and how they are connected to the nucleotide binding There are also cases in which multiple enzymes with the same catalytic domain. activity but different cofactor specificity are found in the same or- The catalytic domains of oxidoreductases are more variable than the ganism. This is the case in barley, which contains two nitrate re- coenzyme binding domains. For example, NAD(P)H-dependent oxi- ductases, regulated in different ways: one of them is specific for NADH doreductases of the D-stereoisomers of 2-hydroxyacids (such as D-gly- (Nar1), while the other can use both NADH and NADPH (Nar7) [65]. cerate dehydrogenase or phosphoglycerate dehydrogenase) have a catalytic domain similar to their well-defined cofactor binding domain, which belongs to the family of Rossmann folds [75]. On the other hand, 3. Structural features of NAD(P)H-dependent oxidoreductases short-chain alcohol dehydrogenases/reductases do not have a distinct, separate catalytic domain to bind the substrate. Instead, they display a The number of NAD(P)H-dependent oxidoreductases for which a single domain consisting of a Rossmann-fold scaffold with a highly structure is available is growing rapidly, allowing protein engineering variable C-terminal extension, which acts as the substrate of these enzymes to be tackled with a rational design or data-driven [76].

330 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

a β-strand 1α-helix β-strand 2 b

NADH

α-helix

β-strand 1

β-strand 2

Fig. 4. Rossmann fold structural motif. A Rossmann fold is typically composed of two beta strands connected by an alpha helix. The loop connecting the first strand with the helix is of variable length and is usually involved in interactions with the cofactor. a. Diagram of the secondary structure topology of the core Rossmann fold. b. NADH bound to a Rossmann fold.

The catalytic domains of enzymes containing Rossmann folds (most three families that can be distinguished based on the size of the sub- of which are oxidoreductases), can belong to seven different super- strate on which they can act, with enzymes of each of the families families of the Structural Classification of Proteins (SCOP) [77]. The sharing structural and mechanistic features. different catalytic domains determine the substrate specificity and the Most members of the alcohol dehydrogenase family of short-chain exact catalytic mechanism employed by the enzyme. NAD(P)H-depen- alcohol dehydrogenases/reductases (SDRs), such as dihydropterin de- dent oxidoreductases also differ in the way that the cofactor binding hydrogenase or 17β-hydroxysteroid dehydrogenase [78], have a key domain and the catalytic domain are connected. Four different types of catalytic Tyr residue whose hydroxyl group donates or accepts a proton connections have been found. The Rossmann domain can be connected from the substrate. An adjacent Lys residue, and the nicotinamide ring to the N terminus of the catalytic domain or to its C terminus; the of NAD(P)+, lower the pKa of the hydroxyl group of the Tyr residue to catalytic domain can be inserted in the Rossmann domain, or the facilitate its role as a proton acceptor/donor (Fig. 5). As the Tyr residue Rossmann domain can be inserted in the catalytic domain [77]. Inter- abstracts a proton from the substrate, a hydride is transferred from the estingly within each of the seven SCOP superfamilies containing Ross- substrate to the oxidized cofactor. However, in certain SDRs able to mann folds, the catalytic domains of each superfamily are always catalyze the reduction of enoyl-thioesters, the Tyr residue is present at connected in the same way to the Rossmann domain. the but does not act as a proton/donor acceptor, or is not present at all (such as in human peroxisomal enoyl-CoA reductase). 4. Classes of NAD(P)H-dependent oxidoreductases and reaction Instead, protons are transferred directly to or from the solvent [76]. mechanisms Medium-chain alcohol dehydrogenases (MDRs) need Zn as a co- factor, and they use either a Tyr-based catalytic mechanism similar to In the following section, we aim to give an overview of the different SDRs, or alternatively a Zn-based mechanism. In the latter, the Zn2+ types of reactions catalyzed by NAD(P)H-dependent oxidoreductases, ion is coordinated, in the absence of the substrate, by a water molecule classified according to the EC system. NAD(P)H-dependent oxidor- and three residues (two Cys and one His) adopting a tetrahedral geo- eductases catalyze a very broad range of redox reactions (Table 2), and metry [79]. After the alcohol substrate binds to the enzyme, it displaces the specific catalytic mechanism differs, even within each EC class. the water molecule from the Zn coordination shell (Fig. 6) and the al- Table 3 shows a simplified summary of the different possible paths cohol substrate transfers a proton to the solvent, forming an alkoxide electrons can follow during the reaction catalyzed by an NAD(P)H-de- intermediate which is stabilized by the catalytic Zn2+ ion. The alkoxide pendent oxidoreductase. Although electron transfer can happen directly ion transfers a hydride to the oxidized cofactor and collapses to an al- between the nicotinamide cofactor and the substrate, there are also dehyde/ketone [79]. many cases where one or more intermediary redox cofactors are in- Finally, long-chain alcohol dehydrogenases (LDRs, such as the volved. It should be noted that while enzymes can potentially catalyze mannitol 2-dehydrogenase of Pseudomonas fluorescens), constitute a redox reactions in both directions, in practice the direction in which the heterogeneous group that differ in the catalytic mechanism and addi- reaction takes place is determined by thermodynamics and the con- tional cofactors needed (Zn, Fe, both, or no metal at all) [80,81]. Many centration of substrates, products and cofactors. Thus, the physiological employ a third catalytic mechanism different to those employed by SDR direction of the reaction might not coincide with the one described here and MDR members, in which a Lys residue accepts a proton from the in all cases. substrate, and then donates it to a solvent molecule. The capacity of the Lys residue to act as a general base is enhanced due to a hydrogen bond 4.1. EC 1.1.1: alcohol dehydrogenases with an Asn residue (Fig. 7). A hydride is then transferred from the substrate to the oxidized cofactor [82]. Alcohol dehydrogenases constitute one of the largest and most di- Iron-containing alcohol dehydrogenases have been less extensively verse groups of NAD(P)H-dependent oxidoreductases, and they can be studied than their Zn-containing counterparts [83]. These enzymes classified into several subfamilies according to different types of cri- contain a divalent Fe2+ ion which is typically coordinated by one Asp teria. Alcohol dehydrogenases catalyze the oxidation of alcohols to the and three His residues arranged in a tetrahedral geometry. The catalytic corresponding aldehydes or ketones, coupled to the reduction of NAD mechanism of Fe-dependent alcohol dehydrogenases is based on a re- (P)+ to NAD(P)H, as well as the reverse reaction. Here, we describe the duction of the pKa of the hydroxyl group of the substrate upon binding

331 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Table 2 Types of reactions catalyzed by NAD(P)H-dependent oxidoreductases. Data was extracted from BRENDA and the recommendations of the Nomenclature Committee of the IUBMB [REF BRENDA + REF EC].

EC Systematic name Reaction 1.1.1 Alcohol dehydrogenases + NAD(P)H + NAD(P)+

1.2.1 + NAD(P)+ Aldehyde dehydrogenases + NAD(P)H

1.3.1 Ene reductases + NAD(P)H + NAD(P)+

1.4.1 Amino acid dehydrogenases + + NAD(P)H + NH3 + NAD(P)

1.5.1 Oxidoreductases of CH-NH + NAD(P)H + NAD(P)+ group

1.6.1 NAD(P)+ transhydrogenases NAD(P)H + NAD(P)+ NAD(P)+ + NAD(P)H

1.7.1 Oxidoreductases acting on Nitrate + NAD(P)H + H Nitrite + NAD(P)H + H20 nitrogenous compounds 1.8.1 Oxidoreductases acting on + NAD(P)H + NAD(P)+ sulfur 1.10.1 Oxidoreductases of diphenols + 2 NAD(P)H + 2 NAD(P)+ and related substances donors

1.11.1 H O + NAD(P)H + H NAD(P)+ + 2 H O Peroxidases 2 2 2 1.12.1 H+ + NAD(P)H H2 + NAD(P)+ Hydrogenases

1.13 and 1.14 + RH + O2 + NAD(P)H-hemoprotein ROH + H2O + NAD(P) - Oxygenases and hemoprotein reductase monooxygenases reductase 1.16.1 M+ + NAD(P)H M + NAD(P)+ Oxidoreductases of metal ions

1.17.1 Oxidoreductases on CH or + NAD(P)H + NAD(P)+ CH2 groups

1.18.1 Oxidoreductases of iron- Oxidized FeS + NAD(P)H Reduced FeS protein + NAD(P)+ sulfur proteins protein donor 1.19 Oxidized + NAD(P)H + H+ Reduced flavodoxin + NAD(P)+ Flavodoxin flavodoxin 1.20.1 Oxidoreductases of + NAD(P)H + NAD(P)+ phosphorous or arsenic compounds 1.21.1 Reductive dehalogenases + NAD(P)H + NAD(P)+ + I-

1.23.1 Pinoresinol + NADPH + H+ Lariciresinol + NAD(P)+ Oxidoreductases reducing C-O-C group Lariciresinol + NADPH + H+ Secoisolariciresinol + NAD(P)+

332 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Table 3 Possible paths of electrons during electron transfer in reactions catalyzed by NAD(P)H-dependent oxidoreductases. Many NAD(P)H-dependent oxidoreductases, including alcohol dehydrogenases, transfer electrons directly between the substrate and the nicotinamide cofactor. However, many others require additional intermediate cofactors, most commonly flavins and metallic centres.

Electron transfer path EC number 1.1.1 1.4.1 1.5.1 Substrate NAD(P)H/NADP+ 1.6.1 1.20.1 1.23.1

Modified NAD(P)H/NADP+ 1.2.1 substrate

1.3.1 1.11.1 Flavin NAD(P)H/NADP+ Substrate 1.14.1 1.16.1 1.21.1 1.7.1 Metallic Substrate redox NAD(P)H/NADP+ 1.8.1 cofactor 1.12.1 1.13.1 Metallic Substrate redox Flavin NAD(P)H/NADP+ 1.17.1 cofactor

Side chain of 1.18.1 Substrate amino acids NAD(P)H/NADP+ 1.19.1

to the enzyme due to interaction with the F2+ ion, which facilitates the 4.2. EC 1.2.1: aldehyde dehydrogenases transfer of a hydride group to NAD+ [84,85]. Fe-dependent alcohol dehydrogenases have been reported to be sensitive to oxygen in- Aldehyde dehydrogenases catalyze the oxidation of aldehydes to activation, unlike Zn-dependent enzymes, due to Fe-catalyzed oxidation carboxylic acids, or in some cases their CoA esters or acyl carrier pro- reactions that can take place in the presence of oxygen [86,87]. tein (ACP) thioesters, coupled to the reduction of NAD(P)+ to NAD(P)

Fig. 5. Active site of human 17β-hydroxysteroid dehydrogenase (PDB code 1A27). 17β-hydroxysteroid dehydrogenase belongs to the SDR family. Substrate is displayed in purple, NADP+ is displayed in grey with heteroatoms coloured independently and residues that are essential for enzyme activity are labelled and coloured dark grey.

333 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Fig. 6. Active site of horse liver dehydrogenase (PDB code 4DXH). Horse liver dehydrogenase belongs to the family of MDR. Substrate is displayed in purple and NAD+ is dis- played in grey with heteroatoms coloured independently. Residues that are part of the coordination shell of Zn2+ are labelled and coloured in dark grey.

H, as well as the reverse reaction. Enzymes of this class are sometimes hydrolysed by a water molecule (which is activated by a Glu residue to referred to by other names, such as carboxylic acid reductases (CAR), carry out hydrolysis), allowing the carboxylic acid to be released depending upon their substrate specificity and/or the physiological [89,90]. direction of the reaction. Some aldehyde dehydrogenases such as methylmalonate-semi- The key catalytic residue of aldehyde dehydrogenases is a Cys re- aldehyde dehydrogenase (MMSDH) yield CoA esters instead of free sidue from whose sulfhydryl group a proton is abstracted by a Glu re- carboxylic acids. These CoA-dependent aldehyde dehydrogenases are sidue, or by a His residue in the exceptional case of Vibrio harveyi al- noteworthy due to their important role in natural and engineered fer- dehyde dehydrogenase (Fig. 8) [88]. The thiolate can act as a mentation pathways which resemble the reverse of beta-oxidation of nucleophile and attack the carbon of the carbonyl group. This nucleo- fatty acids. The E. coli ethanol pathway [91] and the Clostridium butanol philic attack causes the formation of a thiohemiacetal intermediate. The pathway [92] are the best-studied natural examples, but longer-chain thiohemiacetal contains a negatively charged oxygen atom which is alcohols can also be formed in this way [93], and have been the subject stabilized, at least in part, by an Asn residue which is also essential for of recent metabolic engineering studies [94,95]. The CoA-dependent catalysis. The thiohemiacetal oxyanion then spontaneously donates a aldehyde dehydrogenases can occur independently [96] or as part of hydride group to NAD(P)+. In this process, a thioester bond is formed bifunctional aldehyde-alcohol dehydrogenases (ADHE) which are pre- between the aldehyde and the catalytic Cys. The thioester bond is sent in many bacteria [91,92]. Bifunctional aldehyde-alcohol

Fig. 7. Active site of Pseuodomonas fluorescens mannitol dehydrogenase (PDB code 1M2W). Mannitol dehy- drogenase is an example of LDR which does not require a metallic cofactor for its catalytic mechanism. Substrate is displayed in purple, NAD+ is displayed in grey with het- eroatoms coloured independently and residues that are es- sential for enzyme activity are labelled and coloured in dark grey.)

334 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Fig. 8. Active site of human aldehyde dehydrogenase (PDB code 5FHZ). Substrate is displayed in purple, NAD+ is displayed in grey with heteroatoms coloured independently and residues that are essential for enzyme activity are la- belled and coloured in dark grey.)

dehydrogenases are able to catalyze the sequential reduction of a CoA 4.3. EC 1.3.1: ene reductases ester to the aldehyde and then to the alcohol, most commonly acetyl- CoA to acetaldehyde and then to ethanol. They typically comprise two Ene reductases catalyze the hydrogenation of activated alkenes to main domains, an N-terminal aldehyde dehydrogenase domain and an the corresponding alkanes. Only activated alkenes are substrates for iron-dependent alcohol dehydrogenase C-terminal domain. The two these enzymes. For example, one of the carbons participating in the domains are connected by a small linker [97,98]. It is unclear whether double bond should have an electron-withdrawing group (EWG) as a CoA-dependent aldehyde dehydrogenases gained CoA ester formation substituent (a functional group able to remove electron density from the activity during evolution, or if this feature was originally present in double bond, making it more electrophilic). The carbon with the EWG is primitive aldehyde dehydrogenases but it was later lost in most of them referred to as Cα, while the other carbon atom is called Cβ. The reaction [99]. starts with the transfer of a hydride group from a reduced flavin co- Carboxylic acid reductases are able to reduce fatty acids (carboxylic factor to the Cβ, followed by the addition of a proton from a nearby Tyr acids with a long aliphatic chain) to the corresponding aldehydes are of residue to Cα. The proton donated by the Tyr residue is replenished by a special interest due to their ability to generate alcohols and alkanes water molecule. Finally, a reduced NAD(P)H molecule reduces the from the carboxylic acids naturally found in oils and fats. An NADPH- oxidized flavin cofactor [104]. dependent oxidoreductase able to catalyze the reduction of a wide One of the largest groups of ene reductases are the members of the range of fatty acids (with an aliphatic chain containing between 6 and long-known family of Old Yellow Enzymes (OYE). OYE was first iden- 18C atoms) has been identified in Mycobacterium marinum, with the tified in the 1930s as a yellow enzyme able to form a complete re- peculiarity over other aldehyde dehydrogenases that the reaction re- spiratory system together with glucose-6-phosphate dehydrogenase quires ATP in addition to NADPH [100].AnE. coli strain able to convert acting on glucose-6-phosphate and using molecular oxygen as the final fatty acids into long-chain alcohols and alkanes (which have numerous electron acceptor [105]. Since then, variants have been identified in applications as fuels, detergents, food additives and many others) was yeasts, plants and bacteria [106]. When it was purified by Theorell in engineered by introducing the gene encoding the CAR from M. marinum 1935, it was shown to contain a protein, colourless component and a along with an aldehyde reductase (a synonym for alcohol dehy- yellow component essential for activity [107]. Theorell later demon- drogenase) to form alcohols and an aldehyde decarbonylase, a recently- strated that the yellow component was riboflavin 5′-phosphate, also discovered enzyme able to catalyze the conversion of a long-chain al- known as flavin mononucleotide (FMN) [108]. Extensive biochemical dehyde to an alkane and formate [100]. and structural studies have been carried out with OYEs, which have Another type of aldehyde dehydrogenase, known as fatty acyl-CoA/ demonstrated their ability to reduce a large variety of unsaturated ACP reductase, has also been investigated for the ability to produce compounds to their saturated counterparts. However, their physiolo- long-chain aldehydes and alcohols. These enzymes are able to form gical function has not been completely established, although proposals long-chain aldehydes by reduction of fatty acids bound to acyl carrier suggesting a conserved role in the detoxification of electrophilic com- protein (ACP) and/or to CoA, the latter case being similar to the CoA- pounds have been made [106]. dependent aldehyde dehydrogenases described above, but acting on Many of the reactions catalyzed by OYEs are interesting from the long-chain substrates. Some of the enzymes of this group prefer acyl- perspective of an industrial application. For example, OYE can reduce CoA as the substrate, while others act more efficiently on acyl-ACP nitroalkenes to nitroalkanes in a process involving the formation of a substrates, so will generate products from CoA-dependent fatty acid nitronate intermediate. OYE1 from Saccharomyces carlsbergensis with beta-oxidation or ACP-dependent fatty acid synthesis pathways, re- Y196F mutation has been shown to cause the accumulation of the ni- spectively, both of which are being investigated through metabolic tronate intermediate, which could be then chemically alkylated to engineering [101,102]. Interestingly, some of them are able to reduce generate nitroalkanes with two chiral centers [106]. Another potential fatty acid acyl-CoA/ACP directly to long-chain alcohols, requiring oxi- application is to use them to easily carry out small modifications in the dation of two molecules of NAD(P)H, instead of one [103]. structure of drugs to alter their pharmaceutical properties, such as morphine alkaloids. Coexpressing a morphinone reductase (an OYE)

335 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Fig. 9. Active site of E. coli dihydrofolate reductase (PDB code 7DFR). Substrate is displayed in purple, NADP+ is displayed in grey with heteroatoms coloured independently and residues that are essential for enzyme activity are labelled and coloured in dark grey. with a morphine dehydrogenase in E. coli results in a strain able to residue facilitating the role of the Tyr residue by lowering the pKa of efficiently convert morphine and codeine to hydromorphone and hy- the hydroxyl group of the Tyr residue [21]. drocodone, which are effective analgesics [109]. Dihydrofolate reductase (EC 1.5.1.3), which is also essential for the synthesis of folate, is another example of NAD(P)H-dependent oxidor- 4.4. EC 1.4.1: amino acid dehydrogenases eductase acting on CH-NH groups. However, in this case an Asp residue plays the central role in catalysis (Fig. 9). The Asp residue forms a The general reaction catalyzed by amino acid dehydrogenases is the hydrogen bond with the N3 atom of the pterin ring of the substrate and transfer of hydride from the Cα atom of an amino acid to NAD(P)+.Asa a water molecule which establishes another hydrogen bond with the O5 consequence, the corresponding α-keto acid and ammonium are gen- atom. The pKa of the N5 atom of the pterin ring is thus substantially erated. Two amino acid dehydrogenases whose catalytic mechanism elevated, making it able to accept a proton directly from a water mo- has been well studied are glutamate dehydrogenase and phenylalanine lecule. Finally, after the proton has been accepted by the substrate, dehydrogenase [110–112]. In both cases, the catalytic mechanism is hydride transfer from the reduced cofactor takes place [114,115]. very similar. Initially, an Asp residue acts a general base and abstracts a Dihydrofolate reductase is particularly interesting, as it is the target ff proton from the α-amino group of the amino acid. Then, a hydride ion of di erent types of drugs. For example, methotrexate is an inhibitor of is transferred to NAD(P)+ from the Cα, forming an intermediate α- dihydrofolate reductase (DHFR) used as a chemotherapy agent for imino acid [111,113]. Finally, the intermediate imino acid is hydro- several types of cancer. The use of a DHFR inhibitor takes advantage of lyzed to the corresponding α-keto acid and ammonium [111]. the fact that rapidly dividing cells have a very high demand for folate Amino acid dehydrogenases play a key role in the catabolism of since it is necessary to synthesize thymine. Some antibacterial agents fi amino acids, since they allow the removal of their amino group to take aim to speci cally inhibit bacterial DHFR, such as trimethoprim. place, which is a necessary step for their incorporation into other Trimethoprim is used in the standard therapy against the opportunistic catabolic pathways, such as the tricarboxylic acid cycle (TCA cycle). In pathogen Pneumocystis jirovecii. In a recent study, six variants with a the deamination process, an ATP molecule is generated (which can be single amino acid substitution of Pneumocystis jirovecii DHFR were used for energy-requiring processes), and the amino group is released as found to confer resistance against trimethoprim. An experimental drug fl free ammonia, which can then be employed by the cell for different also targeting DHFR but with a higher degree of conformational ex- purposes. Additionally, some organisms use the reverse reaction cata- ibility than trimethoprim (known as OAAG324) was tested against ff lyzed by amino acid dehydrogenases to incorporate free ammonia into these variants, and found to be e ective with one of them [116]. carbon skeletons when the levels of free ammonia are high [20]. 4.6. EC 1.6.1: NAD(P)+ transhydrogenases 4.5. EC 1.5.1: oxidoreductases of CHeNH groups Pyridine nucleotide transhydrogenases catalyze the reversible This family of oxidoreductases comprises a group of enzymatic ac- transfer of hydride between NADH/NAD+ and NADPH/NADP+ pools. tivities acting on a rather large variety of substrates, with the only In most cases, the reaction is as follows: NADPH + NAD+ ⇌ common feature being that all of them contain a single or double CeN NADP+ + NADH. The hydrogen atom is transferred from the C4 bond which donates electrons to reduce NAD(P)+. carbon atom of the reduced cofactor to the C4 carbon atom of the Dihydropteridine reductase (EC 1.5.1.34), a key enzyme for the oxidized cofactor. Two groups of transhydrogenases can be dis- synthesis of folate, catalyzes the reduction of a quinonoid 6,7-dihy- tinguished: energy-linked and non-energy-linked transhydrogenases. dropteridine to the corresponding 5,6,7,8-tetrahydropteridine. NAD(P) Energy-linked transhydrogenases are integral membrane proteins H provides the required electrons, which are accepted by the pyrazine which do not require any flavin cofactor. They receive this name be- heteroaromatic ring. The catalytic mechanism is very similar to that of cause they couple the transhydrogenation reaction to proton translo- the SDR subfamily of alcohol dehydrogenases, with a Tyr residue di- cation across the membrane where they are located. They are found in rectly participating in proton transfer from or to the substrate and a Lys mitochondria, some heterotrophic bacteria and photosynthesizing

336 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347 bacteria [117]. Thanks to the proton gradient generated across both the a molybdenum atom bound to a set of ligands which varies across the mitochondrial inner membrane and the cell membrane of bacteria, the different NRs. Eukaryotic nitrate reductases contain molybdopterin, apparent equilibrium constant of the system is changed, causing a shift while in prokaryotic enzymes the Mo-Co is molybdopterine-guanine towards the production of NADPH and NAD+ [118]. In eukaryotes, dinucleotide. energy-linked transhydrogenase has been suggested to act as a redox Eukaryotic NRs have been extensively studied, and their reaction buffer able to counteract both an excessive depletion of NADPH and a mechanism is well known due to the available structural information. dissipation of the proton gradient. In prokaryotes, the physiological role In eukaryotic NRs, Mo is initially in its Mo4+ state. When nitrate binds is believed to be mostly the generation of NADPH for anabolism [4]. to the active site, it forms the reaction intermediate by coordinating the These transhydrogenases are also called Re/Si-specific transhy- Mo atom with one of its O atoms, displacing a water molecule which drogenases or AB transhydrogenases, as they transfer hydrogen 4A of was previously bound to Mo. The formation of the reaction inter- the nicotinamide ring (situated on its Re face) from NADH to NADP+, mediate causes Mo electrons to shift towards the created MoeO bond. or hydrogen 4B (situated on the Si face) from NADPH to NAD+. As a consequence, Mo is oxidized to Mo6+ and the bond between Mo Non energy-linked transhydrogenases are soluble flavoproteins and the coordinating O atom from nitrate becomes a double bond. This which contain FAD. They are less widespread than energy-linked causes the bond between this O atom and nitrate to break, forming transhydrogenases and only found in some heterotrophic bacteria. nitrite which is subsequently released. Finally, transfer of two electrons Usually, only one of the two types of transhydrogenases is found in a and two protons from NAD(P)H via a cytochrome b5 domain cause specific organism, with the exception of Enterobacteriaceae (including regeneration of Mo4+ and reduction of the O atom bound by a double Escherichia coli), which possess one energy-linked and one non energy- bond to a water ligand [125]. linked transhydrogenase encoded by the pntAB and udhA re- spectively. Non energy-linked transhydrogenases are also known as Si/ 4.8. EC 1.8.1: oxidoreductases acting on a sulfur group Si-specific transhydrogenases or BB transhydrogenases, since they transfer hydrogen 4B of the nicotinamide ring, both from NADH to This group also consists of a diverse set of oxidoreductases whose NADP+ and from NADPH to NAD+. Contrary to energy-linked trans- only common characteristic is that they act on a sulfur-containing hydrogenases, it has been suggested that the physiological role of non- group of their substrate, which can be of very different types. These energy-linked transhydrogenases is to convert NADPH to NADH [4]. include both inorganic forms of sulfur (such as sulfite and sulfide), and However, this seems to be dependent on growth conditions and or- a variety of sulfur-containing organic molecules (including , ganism, with some reports indicating that non energy-linked transhy- glutathione, coenzyme A and many others). As in the case of enzymes drogenases can actually increase NADPH availability when the demand belonging to EC 1.7.1, the role of NAD(P)H is to provide electrons to for NADPH is increased [119]. Interestingly, it has been found that regenerate the other cofactors directly involved in the catalysis. expressing a non-energy-linked transhydrogenase in Synechococcus re- NADPH-dependent sulfite reductase (EC 1.8.1.2), such as E. coli sults in a reduced growth rate [120], perhaps reflecting the importance sulfite reductase, catalyzes the reduction of sulfite to sulfide by trans- of the highly reduced state of the NADPH pool normally maintained in ferring electrons provided by NADPH, one of the key steps of the as- cyanobacteria. similation of inorganic sulfur into organic molecules. This enzyme Both types of transhydrogenases have been explored as metabolic shares structural and mechanistic features with nitrite reductase, in- engineering targets to improve the yield of products whose synthesis cluding the presence of a sirohaem cofactor and a [4Fe-4S] cluster. depends on cofactor availability. Results have been mixed, with some Additionally, both of them catalyze the transfer of six electrons (which studies showing that overexpressing pntAB is sufficient to enhance leads to the formation of ammonia in the case of nitrite reductase). In NADPH regeneration [121] while others show that the overexpression fact, purified sulfite reductase is also able to catalyze the reduction of of pntAB does not increase NADPH availability and can even decrease it nitrite to ammonia [126,127]. The reaction mechanism involves three [122]. In the latter study, it was found that overexpressing udhA in- successive transfers of two electrons, provided by NADPH, and protons creased the levels of available NADPH. to the oxygen atoms of sulfite, which binds as an axial ligand of the sirohaem Fe atom [29]. 4.7. EC 1.7.1: oxidoreductases acting on other nitrogenous compounds (other than CHeNH groups) 4.9. EC 1.10.1: dehydrogenases of diphenols

This group includes dehydrogenases acting on a variety of different EC class 1.10 includes all oxidoreductases able to act on diphenols compounds containing nitrogen and which cannot be classified into any and related products. There are five subtypes this EC class, which vary of the other categories of oxidoreductases. Substrates include both in- in the redox partner they use to transfer the electrons from the diphenol organic compounds (such as nitrate and nitrite) and organic compounds substrate: NAD(P)+, a cytochrome, oxygen, a quinone, or a copper (such as azobenzene and nitroquinoline). The exact reaction mechan- protein. Those using oxygen as the electron acceptor comprise by far isms of many of these enzymes are still poorly understood, partly due to the highest diversity of enzymatic activities, while there is only one the lack of structural information for many of them, although for an known enzymatic activity of such type dependent on NAD(P)+: trans- example see [123]. However, in most of the cases NAD(P)H does not acenaphthene-1,2-diol dehydrogenase (EC 1.10.1.1), which oxidizes directly transfer electrons to the substrate, but instead provides the said substrate to acenaphthenequinone. This NAD(P)H-dependent en- electrons to regenerate the reduced form of the other cofactor which zyme was identified and purified in 1973 from rat (and other mam- directly participates in the reduction of the substrate. malian) liver extracts [32]. However, the structure of the enzyme has NAD(P)H-dependent nitrate reductases (EC 1.7.1.1–3) are relatively not been solved, and no information about the reaction mechanism is well characterized, and they catalyze the reduction of nitrate to nitrite, available. with NAD(P)H providing the source of electrons for the reduction of nitrogen. There are four different types of nitrate reductases (NRs): 4.10. EC 1.11.1: peroxidases eukaryotic assimilatory NR, cytoplasmic bacterial assimilatory NR, membrane-bound bacterial respiratory NR and periplasmic bacterial Peroxidases catalyze the transfer of electrons from an electron donor dissimilatory NR [124]. Only assimilatory nitrate reductases, which to H2O2. Both NADH-dependent and NADPH-dependent peroxidases carry out the incorporation of nitrogen into organic molecules for have been found (EC 1.11.1.1 and EC 1.11.1.2 respectively). The re- + + growth, are directly NAD(P)H-dependent (nitrate and periplasmic ni- action they catalyze is NAD(P)H + H +H2O2 ⇌ NAD(P) +2H2O. trate reductases). All NRs contain a molybdenum cofactor (Mo-Co) with NADH has been well characterized, and its structure and

337 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Fig. 10. Active site of Enterococcus faecalis NADH-dependent peroxidase (PDB code 2NPX). NAD+ and FAD are displayed in grey and light blue respectively with heteroatoms coloured independently and residues that are essential for enzyme activity are labelled and coloured in dark grey. mechanism long known. In addition to NADH, the catalytic centre of S) cluster relay. the enzyme includes FAD, a Cys residue and a His residue (Fig. 10). The The active site of [NiFe]-hydrogenases is ligated by four Cys re- Cys residue is adjacent to the FAD cofactor. As a first step, NADH binds sidues. Two of the Cys are bridging ligands of both metal ions, while the to the enzyme with FAD in its oxidized state. The binding of NADH other two are bound to the Ni atom alone [129]. The Fe atom binds − causes the electron density of the isoalloxazine group of FAD to shift three diatomic ligands [130], two CN ligands and one CO ligand towards the Cys residue. Then, H2O2 binds to the enzyme forming hy- [131] (Fig. 11). The bridging ligand between the metal ions is either a drogen bonds with the Cys and His residues, and other amino acids hydroxo or a hydro-peroxide depending on the redox state of the active close to the active site. The Cys residue performs a nucleophilic attack site [132]. − on H2O2, cleaving the OeO bond to yield an OH anion and a sulfenic The catalytic core of [FeFe]-hydrogenases consists of four domains acid derivative. The hydroxide anion abstracts a proton from the His [133], which help with electron transfer to and from the active site and residue, and is released as a water molecule. The sulfenic acid deriva- cofactors. The active site of [FeFe]-hydrogenases consists of a 4Fe-4S tive is reduced by the transfer of two electrons and a proton from NADH cluster ligated by four Cys, which is coordinated via a single conserved through FAD, leading to the formation of a second water molecule Cys thiolate sulfur atom to the two Fe core. Each of those two iron ions − which is released together with NAD+. Finally, the His residue receives is attached to a CO and CN ligand and a bridging CO ligand [134]. a proton from the solvent, to regenerate its initial protonated state [33]. A small number of NAD(P)H-dependent hydrogenases have been Another class of NAD(P)H dependent peroxidases catalyzes the re- described. All of the NAD(P)H-dependent hydrogenases are multimeric duction of hydroperoxy derivatives of fatty acids to the corresponding enzyme complexes [135], including a subunit with a flavin moiety hydroxy acids (EC 1.11.1.22). The enzyme was originally found in which carries out the oxidation/reduction reaction of the nicotinamide Synechocystis PCC 6803 [128], and uses electrons from NADPH to re- cofactor. The best studied member is the bidirectional soluble [NiFe]- duce peroxidized lipids to hydroxy acids. These enzymes share se- hydrogenase of Cupriavidus necator H16 [136], which oxidizes or pro- quence similarity to glutathione peroxidases, but no catalytic activity duces H2 in response to changes in cytoplasmic reduction states. can be detected when reduced glutathione is provided as the electron donor. One of the major differences with glutathione peroxidases is the 4.12. EC 1.13 and 1.14: oxygenases and monooxygenases absence of a residue, which is replaced by a standard Cys residue. This type of NADPH-dependent peroxidases has been hy- Monooxygenases catalyze the transfer of a single atom of oxygen to pothesized to serve as a defense system against hydroperoxide deriva- a substrate, requiring first that the oxygen molecule be activated, which tives of unsaturated fatty acids, which can lead to oxidative damage takes place by transferring electrons to the oxygen molecule. Depending [128]. on the type of monooxygenase, electrons can be donated either from the substrate or from a cofactor, which determines the type of reactive

4.11. EC 1.12.1: oxidoreductases of H2 (hydrogenases) oxygen intermediate. Here, we focus on NAD(P)H-dependent mono- oxygenases. Hydrogenases catalyze the oxidation of molecular hydrogen or re- Cytochrome P450 are haem B monooxygenases (CYPs), which are duction of protons and are classified into three main phylogenetically able to catalyze a wide range of reactions (such as epoxidations, hy- distinct classes, based on the metals found at their active site: [NiFe]- droxylations, heteroatom-dealkylations/oxidations, dehalogenations, hydrogenases, [FeFe]-hydrogenases, and the lesser-studied [Fe-only]- dehydrations, reductions, dehalogenations and oxidative deamina- hydrogenases. In all cases, the catalytic mechanism of hydrogen oxi- tions). The haem group activates molecular oxygen by transferring dation or proton reduction requires electron transfer to and from the electrons to it, which in the case of NAD(P)H-dependent CYPs are ul- active-site metals, which are directly responsible for the oxidation of timately provided by NAD(P)H. The mechanism by which the electrons hydrogen or reduction of protons. The electrons can be transferred to or are transferred to the haem group from the nicotinamide cofactor varies from a variety of donors/acceptors, which can include NAD(P)H/NAD between different CYPs. (P)+. In all cases, the transfer of electrons occurs via an iron sulfur (Fe- The general mechanism of NAD(P)H-dependent CYPs can be divided

338 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Fig. 11. Active site of Hydrogenophilus thermoluteolus [NiFe]-hydrogenase (PDB code 5XF9). Ni3+ is displayed in green and Fe2+ is displayed in orange. Ligands of the co- ordination shell of both ions are labelled and coloured.

into four main steps. Firstly, the substrate binds to the enzyme, and an water molecule and the formation of the oxidized state of the flavin. electron is transferred to the haem group from NAD(P)H via a flavin- The role of the NAD(P)H is to restore the reduced form of the flavin in containing cytochrome P450 reductase, to reduce Fe3+ to Fe2+. the catalytic site [37,140]. Secondly, an oxygen molecule binds to the complex, forming a complex There are also some NAD(P)H-dependent monooxygenases where known as oxy-P450. Thirdly, the transfer of a second electron and two the cofactor that activates O2 is not a haem group or a flavin, but other protons to the oxy-P450 complex causes the cleavage of the OeO bond, redox cofactors. An example is methane monooxygenase (MMO), which leading to the formation of a reactive intermediate and a water mole- catalyzes the incorporation of an oxygen atom into methane (it is also cule. Then, the reactive intermediate inserts the oxygen atom into the active with other hydrocarbons), yielding methanol, which is the cri- substrate. Finally, product dissociation regenerates the initial state of tical first step in methanotrophic carbon assimilation. In MMO, acti- the enzyme. In this state, a molecule of water coordinates the Fe atom vation of molecular oxygen is carried out by a metallic cofactor con- of the haem-group as an axial ligand. The other ligands of the Fe atom sisting of two Fe ions each coordinated by six ligands (a combination of are the nitrogen atoms of the haem group and a Cys residue present in His and Glu residues and water molecules) [141]. After one oxygen the active site of all CYPs, which makes it more reactive [137]. The atom is transferred to the substrate, the reduced form of the metallic effect of the Cys ligand on reactivity is due to two factors, it lowers the centre is regenerated by the transfer of two electrons from NAD(P)H reduction potential of the active site, and it increases the pKa of the [142]. There are two types of MMO, soluble MMO (sMMO) and mem- oxygen-bound intermediate, which facilitates its protonation, resulting brane-bound particulate MMO (pMMO). Soluble MMO is dependent on in the cleavage of the OeO bond [138]. NAD(P)H, and contains three subunits: MmoA, MmoB and MmoC. In most CYP systems, the cytochrome P450 component and the re- MmoA carries out the oxygenation reaction, while MmoC binds NAD(P) ductase component, which transfers the electrons to the cytochrome H and provides to MmoA the electrons required for the reaction through P450 from the electron donor, are separate proteins. However, some MmoB [143]. MMO has been extensively studied thanks to its ability to simpler systems have been found in which both components are fused oxidize a variety of hydrocarbons under mild conditions, and carry out in a single polypeptide chain, making heterologous expression and a gas-to-liquid transformation for natural gas (methane to methanol). protein engineering more facile. An example of this is the well-char- However, protein engineering studies have been hindered by the fact acterized BM3 enzyme from Bacillus megaterium, which is a soluble that no functional MMO has been expressed in E. coli [144]. enzyme able to hydroxylate fatty acids of varying length between 12 and 18 carbon atoms, as well as the corresponding amides and alcohols. 4.13. EC 1.16.1: oxidoreductases of metal ions BM3 also displays a weak hydroxylation activity towards shorter chain alkanes, and has been modified, by means of random mutagenesis, to This class of enzymes comprises NAD(P)H-dependent oxidor- obtain several variants with enhanced activity towards octane, being up eductases able to transfer electrons to or from metallic ions. The me- to five times more efficient than the wild type enzyme. One of the tallic substrate can be in a free form (as in the case of mercury re- variants contained a single point mutation of a Glu residue in the sur- ductase), chelated by relatively small molecules (for example, ferric- face of the protein by a His residue. The effect of this mutation was chelate reductase or aquacobalamin reductase) or forming part of an- surprising, given the fact that this residue is located relatively far from other protein (such as methionine synthase reductase and transferrin the haem group [139]. reductase). Flavin-dependent monooxygenases catalyze a very wide range of In general, when NAD(P)H-dependent oxidoreductases of metal ions oxidative reactions, and use either FMN or FAD as cofactors. In order to act to reduce the metal, they first transfer the electrons from NAD(P)H introduce an oxygen atom to the substrate, oxygen first binds to the to FAD, and then the reduced FAD donates the electrons to the metallic flavin in its reduced form, to form peroxyflavin or hydroperoxyflavin. ion. This electron transfer path allows the enzyme to transfer the Then, the flavin derivative performs a nucleophilic or an electrophilic electrons provided by NAD(P)H to the metallic atoms one at a time, as attack, depending on the protonation state of the flavin derivative, in most cases they require the transfer of one single electron in order to which leads to the oxygenation of the substrate, the production of one be reduced or oxidized. Mercuric reductase and cyanocobalamin

339 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Fig. 12. Active site of Pseudomonas aeruginosa NADPH-dependent mercuric reductase (PDB code 4K7Z). Substrate is displayed in purple, NADP+ and FAD are displayed in grey and light blue respectively with heteroatoms coloured independently and the that coordinate Hg2+, essential for enzyme activity, are labelled and coloured in dark grey. reductase are exceptions, as they perform two-electron reduction of Leucoanthocyanidin reductase (EC 1.17.1.3), which catalyzes the Hg2+ to Hg0 and Co3+ to Co+, respectively, with FAD as a cofactor. reduction of 2R,3S,4S-flavan-3,4-diols to 2R,3S-flavan-3-ols (for ex- Human methionine synthase reductase, which reduces the Co2+ atom ample, leucocyanidin to catechin) coupled to the oxidation of NADPH of the cobalamin cofactor found in methionine synthase, follows a to NADP+, uses a different mechanism. Firstly, a step of dehydration of variation of this path where electrons are first transferred from NADPH the substrate takes place. In order to do so, a Lys residue deprotonates to FAD, then to FMN and finally to the metal [145]. the phenolic O7 atom of the substrate. This makes the hydroxyl group Mercuric reductase is an enzyme of particular interest for phytor- of C4 act as a leaving group which takes a proton from the adjacent O5. emediation, since it could potentially be used to generate plants capable The O5 atom recovers the proton from a His residue. This generates a of detoxifying mercuric compounds. Many soil and enteric bacteria quinone methide intermediate, to which a hydride is transferred di- have a detoxification system for organomercuric compounds where rectly from NADPH to generate catechin and NADP+ [44]. mercuric reductase plays a key role. This system allows these organisms to generate volatile mercury (Hg0) from ionic mercury (Hg2+)bya two-electron reduction, thereby avoiding toxicity associated with the 4.15. EC 1.18.1 and 1.19.1: oxidoreductases of iron-sulfur proteins and of tendency of Hg2+ to cause sulfhydryl chelation [146]. The first step of flavodoxin the reaction is the binding of Hg2+ to the active site of the enzyme, where it is chelated by the thiolate groups of two deprotonated Cys This group of oxidoreductases are able to catalyze the reduction and residues. Then, two electrons are transferred from NADPH to FAD. Fi- oxidation of the Fe-S clusters of iron-sulfur proteins using NAD(P)H/ + nally, the reduced FAD reduces the Cys-Hg2+-Cys complex, and the NAD(P) as the electron donor/acceptor. The iron-sulfur proteins on resulting Hg0 is released [147] (Fig. 12). which they can act include ferredoxin, adrenodoxin, putidaredoxin and rubredoxin, with the latter usually being classified as an iron-sulfur protein even though it does not contain a canonical Fe-S cluster but

4.14. EC 1.17.1: oxidoreductases acting on CH or CH2 groups instead contains a lone iron ion coordinated by several Cys residues. Iron-sulfur proteins can have very different redox potentials, and can These oxidoreductases catalyze the oxidation or reduction of CH or thus participate in a variety of redox reactions. Consequently, the

CH2 groups, which in most cases results in the addition or removal of a physiological role they play is quite varied, including participating as hydroxy group. The only exception is xanthine dehydrogenase, which electron carriers in photosynthesis [149], donating electrons to cyto- catalyzes the addition of an oxo group. chrome P450 for steroid biosynthesis [150] or providing electrons to Xanthine dehydrogenase (EC 1.7.1.4) and nicotinate dehydrogenase carry out hydroxylation reactions during ω-oxidation of fatty acids (EC 1.7.1.5), which catalyze respectively the oxidation of xanthine to [151]. All NAD(P)H-dependent oxidoreductases of iron-sulfur proteins urate with NAD+ as the electron acceptor and the oxidation of nicoti- require an FAD cofactor, which acts as an intermediary in the transfer nate to 6-hydroxynicotinate with NADP+, employ similar reaction of electrons. This is due to the fact that iron-sulfur proteins are one- mechanisms. Both contain a molybdenum cofactor (molybdopterin), electron donors/acceptors, while NAD(P)H/NAD(P)+ only participate two Fe-S clusters of the [2Fe-2S] type and FAD [148]. All of these redox in two-electron transfers. Since FAD can participate both in one-elec- centres are positioned linearly, and close enough to allow sequential tron transfers and two-electron transfers, it can first receive two elec- transfer of electrons through them. After substrate binding, an OH li- trons from NAD(P)H to be reduced to its hydroquinone fully reduced e gand of the Mo is deprotonated by a Glu residue. The resulting MoeO form, and then transfer them one at a time to the substrate, being performs a nucleophilic attack on the carbon atom to be hydroxylated oxidized first to a semiquinone form and finally to the fully oxidized and a hydride group is transferred to the Mo cofactor, reducing Mo6+ quinone form. Structures of these enzymes show FAD and NAD(P)H/ to Mo4+. Then, the bond between the Mo cofactor and the substrate is NAD(P)+ are close to each other, suggesting direct electron transfer hydrolysed, releasing the free hydroxylated product. Finally, the hy- between both redox cofactors (Fig. 14). The enzymes differ on the route dride groups are transferred first to the Fe-S centers, then to FAD and followed by electrons from the Fe-S cluster to FAD, with some of them eventually to NAD(P)+ to yield NAD(P)H [148] (Fig. 13). involving several intermediate residue side chains [152]. Additionally,

340 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Fig. 13. Overview of cofactors involved in electron transfer in bovine milk xanthine dehydrogenase (PDB code 3BDJ and 3UNA). Oxopurinol, an inhibitor that binds to the same site than the substrate, is displayed in purple; molybdopterine, FAD and NAD+ are displayed in dark green, blue and grey respectively; heteroatoms are coloured individually.

the NADP+-dependent ferredoxin oxidoreductase of some organisms to NAD+, generating NADH. are also able to catalyze redox reactions with flavodoxin (EC 1.19.1.1), The exact mechanism through which PTDH catalyzes the reduction another small, FMN-containing protein which participates in several of phosphite remains unclear, but there are some proposed reaction redox reactions and can replace ferredoxin in some of its functions mechanisms based on the high degree of between under low iron availability conditions [153]. This is the case, for ex- PTDH and 2-D-hydroxyacid dehydrogenases. There are three residues ample, of E. coli and Anabaena ferredoxin-NADP+ reductase [49]. essential for catalysis: a His, a Glu and an Arg (Fig. 15). The His residue has been proposed to act as a general base to abstract a proton from a 4.16. EC 1.20.1: oxidoreductases of phosphorus or arsenic compounds water molecule, generating a hydroxide ion. The hydroxide ion then carries out a nucleophilic attack on phosphite, and at the same time a Phosphonate dehydrogenase (EC 1.20.1.1), also called phosphite hydride leaving group is transferred to NAD+. The Glu residue probably dehydrogenase (PTDH), catalyzes the oxidation of phosphonates or helps to orient the His residue for catalysis and modulates its pKa to phosphite to phosphates. During the reaction, electrons are transferred facilitate its role as a general base. Finally, the Arg residue is believed to

Fig. 14. Interface of the complex between maize ferredoxin and ferredoxin reductase (PDB code 1GAQ and 1GJR). Ferredoxin (the substrate of ferredoxin reductase) is displayed in purple; NADP and FAD are displayed in grey and blue respectively; heteroatoms are coloured individually.

341 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

Fig. 15. Active site of Pseudomonas stutzeri phosphite de- hydrogenase (PDB code 4E5K). Sulfite, an inhibitor of the enzyme which binds in the same way than the substrate does, is displayed in purple; NAD+ is displayed in grey with heteroatoms coloured independently and residues that are essential for enzyme activity are labelled and coloured in dark grey.

be essential for binding the substrate and correctly orienting it. Muta- a leaving group. However, this has not been confirmed. It is also unclear tions in any of these residues have been found to compromise catalytic whether the hydride group would be transferred directly from NADPH efficiency [154,155]. or from an intermediate species [159].

4.17. EC 1.21.1: reductive dehalogenases 4.18. EC 1.23.1: oxidoreductases reducing CeOeC group

There are two enzymes classified as NAD(P)H-dependent reductive Pinoresinol-lariciresinol reductase (EC 1.23.1.1–4) is a multi- dehalogenases: iodotyrosine and 2,4-dichlorobenzoyl-CoA functional enzyme found in certain plants (such as Forsythia and Thuja) reductase. able to catalyze the reduction of pinoresinol to lariciresinol and of Iodotyrosine deiodinase catalyzes the reductive dehalogenation of lariciresinol to secoisolariciresinol, with NADPH providing the required mono- and side-products generated during the bio- electrons. This enzyme is unique in its ability to directly reduce an ether synthesis of thyroxine (the prohormone of triiodothyronine, the most group to an alcohol. The crystal structure of the enzyme from Thuja active hormone). The reaction involves the removal of the io- plicata has been determined, and shows that the NADPH and substrate dine substituents from the derivative and their reduction to binding pockets are close enough for direct electron transfer to occur. iodide ions, coupled to the oxidation of NADPH to NADP+. This process The exact mechanism and reaction intermediates through which cata- allows the scavenging of iodide, which can then be used for the lysis proceeds are still unknown due to the lack of published structures synthesis of more or other processes. Defects in this of enzymes with substrate bound [53]. enzyme can lead to iodide deficiency, which can have several detri- mental effects to health such as . The mechanism em- 5. Metabolic and protein engineering of NAD(P)H-dependent ployed by iodotyrosine deiodinase has not still been fully established, oxidoreductases although recent structures of the enzyme bound to its substrate have provided some insight [51,156]. The enzyme contains an FMN cofactor 5.1. Metabolic engineering which plays a key role in the catalytic process by carrying out a step- wise reduction of the substrate by means of sequential one-electron There have been numerous metabolic engineering studies in which transfers and not through a single two-electron transfer as previously the metabolic network of an organism has been altered by the addition proposed [51,157,158]. of NAD(P)H-dependent oxidoreductase(s), or pathways including them 2,4-Dichlorobenzoyl-CoA reductase was identified in [100,160–164]. One of the most frequent goals of this type of approach Corynebacterium sepedonicum, an organism able to grow with 2,4-di- is the efficient production of a given chemical of interest without the chlorobenzoate as the sole source of energy and carbon, which could be need for chemical synthesis or purifying enzymes, and ideally from of potential interest for the remediation of sites contaminated by renewable feedstocks and with a lower energy consumption due to the polychlorinated biphenyl [159]. In order to catabolise 2,4-di- mild conditions at which reactions can be catalyzed by NAD(P)H-de- chlorobenzoate, it is first converted to the corresponding CoA thioester, pendent oxidoreductases. However, metabolic engineering provides a 2,4-dichlorobenzoyl-CoA. The thioester is then subject to a reductive useful tool not only for this purpose, but also others such as improved dehalogenation catalyzed by 2,4-dichlorobenzoyl-CoA reductase, where biodegradation [165,166]. This is usually achieved by genetically ma- NADPH provides the electrons. This reaction leads to the production of nipulating a candidate organism to add, modify (commonly by over- 4-chlorobenzoyl-CoA, which undergoes an additional dehalogenation expression) and/or remove the genes coding for one or more enzymes. (independent of NAD(P)H) before the thioester bond is hydrolyzed. An example of successful metabolic engineering leading to the de- Subsequent hydroxylations and oxygenations complete the catabolic velopment of a synthesis process suitable for commercial application pathway. The reaction mechanism of 2,4-dichlorobenzoyl-CoA re- was the construction of an E. coli strain able to produce 1,3-propanediol ductase has been speculated to involve a nucleophilic attack of the C2 (a precursor for the generation of several polymers, including fibers) at position of the substrate by a hydride group, with the Cl atom acting as high titer when growing aerobically and using only glucose as the

342 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347 substrate [167]. Several additions and deletions of genes of E. coli K12 engineered an NADH-dependent methylglyoxal reductase from were carried out to divert carbon from dihydroxyacetone phosphate Saccharomyces cerevisiae (GRE2) to have enhanced aldehyde reductase (DHAP), generated during glycolysis, to 1,3-propanediol. Genes en- activity towards furfural and 5-hydroxymethylfurfural [170]. From a coding glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate library of variants generated by means of error-prone PCR, two variants phosphatase from Saccharomyces cerevisiae were introduced in the ori- with the enhanced desired activity were identified. Interestingly, one of ginal strain, which allow the generation of glycerol from DHAP. Ad- these variants also acquired the ability to use NADPH as a cofactor in ditionally, genes encoding glycerol dehydratase from Klebsiella pneu- addition to NADH. In another study, Li et al. aimed to improve the 3- moniae were also introduced, which catalyze the conversion of glycerol hydroxypropionaldehyde (3-HPA) reductase activity of a 1,3-propane- to 3-hydroxypropionaldehyde. The final step of reduction to 1,3-pro- diol oxidoreductase (YqhD) with the goal of obtaining an efficient en- panediol was found to be carried out by the endogenous oxidoreductase zyme for the biological production of 1,3-propanediol [167]. After yqhD. In order to improve the 1,3-propanediol yield, genes encoding screening a library of mutants generated through error-prone PCR, two glycerol kinase and glycerol dehydrogenase were deleted. This pre- variants with improved kinetic properties were isolated. They displayed vented glycerol from being redirected to the main glycolytic pathway. both a lower Km and a higher kcat towards 3-HPA. In a more recent study, an E. coli strain able to produce 1,4-buta- There have also been multiple attempts to modify the specificity of nediol (BDO) was generated. This study combined addition, removal NAD(P)H-dependent oxidoreductases for the cofactor they use for the and modification of enzymes, showing how different approaches can be electron transfer reaction, as controlling the cofactor specificity of this combined to achieve optimal results [168]. Firstly, a set of candidate class of enzymes can be used to optimally engineer the cellular meta- pathways for the synthesis of BDO from several central metabolites was bolism by achieving a better balance of cofactor availability [171,172]. computationally identified and ranked according to several criteria, During the last two decades, there have been a considerable number of such as thermodynamic parameters, pathway length or number of non- successful cases of relaxation or even reversal of NADH/NADPH co- native steps. A pathway to generate BDO from succinate was chosen factor specificity. Altering an NAD(P)H-dependent oxidoreductase to and implemented. The pathway involved six reactions, catalyzed by use NADH/NAD+ instead is of great interest for non-cell-based in- succinyl-CoA synthetase, CoA-dependent succinate semialdehyde de- dustrial applications since the phosphorylated cofactors are sig- hydrogenase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl- nificantly more expensive and less stable than their nonphosphorylated CoA , 4-hydroxybutyryl-CoA reductase and an alcohol de- counterparts, making the enzymes that are dependent on NADPH/ hydrogenase able to reduce 4-hydroxybutyraldehyde to BDO. All of the NADP+ less attractive for industrial-scale applications [173–175]. Ex- enzymes except succinyl-CoA synthetase and 4-hydroxybutyryl-CoA amples of this include an NADPH-specific xylose reductase from Can- transferase were NAD(P)H-dependent oxidoreductases. An additional dida boidinii which was engineered to be able to use also NADH by gene containing α-ketoglutarate decarboxylase was also added, which means of computational design through minimization of the binding allowed the conversion of α-ketoglutarate into one of the intermediates energy of the cofactor in variants were the residues of the cofactor of the pathway. In order to increase the BDO yield, the genes coding binding pocket were mutated. A set of 10 candidate variants were certain enzymes were deleted to divert carbon and energy towards its identified, which were experimentally tested, leading to seven variants production: E. coli alcohol dehydrogenase, pyruvate formate , able to reduce xylose with NADH [176]. In general, cofactor specificity lactate dehydrogenase and malate dehydrogenase. Finally, protein en- reversal must be done on a case-by-case basis. However, there have gineering was used to introduce a R163L mutation in citrate synthetase, been some attempts at developing general methodologies for specific which reduced the inhibition of this enzyme by NADH. This improved subsets of NAD(P)H-dependent oxidoreductases. For example, Brink- the TCA-cycle flux, enhancing the production of α-ketoglutarate. The mann-Chen et al. managed to develop a general approach to reverse the resulting strain was able to produce relatively high titers of BDO when cofactor specificity of ketol-acid reductoisomerases from NADPH to grown microaerobically in M9 medium supplemented with glucose, NADH combining information derived from crystal structures and xylose, sucrose or mixed sugars. multiple sequence alignments. By applying this approach, they ob- Another interesting possibility is engineering cells to modify the tained variants for several enzymes of this class whose catalytic effi- balance of reduced and oxidized cofactors, such as NADH/NAD+. This ciency with NADH was comparable to that of the wild type enzymes could further optimize the production of metabolites when the limiting with NADPH or even higher [177]. This is noteworthy, as other studies factor becomes the availability of a specific form of the cofactor. attempting to change the cofactor specificity of an NAD(P)H-dependent Berríos-Rivera et al. managed to achieve an increased NADH/NAD+ oxidoreductase typically report engineered variants which do not reach ratio in E. coli by substituting the native, cofactor-independent formate a catalytic efficiency with the new cofactor as high as that of the ori- dehydrogenase with a NADH-dependent formate dehydrogenase. ginal enzyme with its native cofactor. The opposite cofactor specificity Interestingly, the increased ratio of NADH/NAD+ not only caused an reversal from NADH/NAD+ to NADPH/NADP+ has also been reported. increased production of reduced fermentative products under anaerobic This type of reversal has been applied to generate enzymes suitable to conditions (the ethanol-acetate ratio was greatly increased), but also an be used in systems where regeneration of NADPH is necessary. With activation of fermentative pathways even under aerobic conditions this purpose, Lerchner et al. engineered the highly NADH-specific ala- [169]. nine dehydrogenase from Bacillus subtilis to use NADPH with nearly the same catalytic efficiency that the native enzyme has with NADH. This 5.2. Protein engineering allowed them to couple the amino acid dehydrogenase to an alcohol dehydrogenase and an aminotransferase to efficiently produce dicyclic Various properties of NAD(P)H-dependent oxidoreductases have diamines from the corresponding dicyclic dialcohols. In order to obtain been targeted by protein engineering and/or directed evolution ap- this variant, homology models of both the Bacillus subtilis enzyme and proaches to make them more suitable for applications. These include alanine dehydrogenase of Shewanella, which was known to be able to enhancing the kinetic properties of enzymes, increasing their stability use both NADH and NADPH as cofactors, were generated based on the and modifying the specificity of enzymes for their substrate or cofactor. crystal structure of Mycobacterium tuberculosis alanine dehydrogenase The improvement of kinetic properties is of interest not only for [178]. Then, mutations were introduced in the Bacillus subtilis enzyme improving the natural reaction of a given oxidoreductase, but also when to mimic the cofactor binding pocket of the Shewanella enzyme [179]. an enzyme with a broad substrate specificity is available but the activity Another frequent goal when engineering oxidoreductases is the towards a specific substrate of interest is low. There are a few cases of modification of substrate specificity. There are several examples, often oxidoreductases whose kinetic properties for either the main substrate concerning alcohol dehydrogenases, where variants with activity to- or secondary substrates have been improved. For example, Moon & Liu wards a new substrate or a different isomer of one of the natural

343 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347 substrates were generated. For example, Laadan et al. identified a substrates to catalyze electron transfer reactions. In recent years there mutant of alcohol dehydrogenase 1 (ADH1) of Saccharomyces cerevisiae have been a growing number of studies where novel applications based able to catalyze the NADH-dependent reduction of 5-hydro- on these enzymes are proposed, or new methods proposed where these xymethylfurfural to 2,5-bis-hydroxymethylfuran, which could enhance enzymes or organisms containing them can replace other less ad- the production of bioethanol through fermentation of lignocellulosic vantageous methods for catalysis. substrates by Saccharomyces cerevisiae. In order to do so, Saccharomyces Various factors underpin ongoing progress in research towards ap- cerevisiae cells where the ADH1 gene had been deleted were trans- plications based on NAD(P)H-dependent oxidoreductases. The ex- formed with the library of variants and selected by their ability to grow plosive growth in available genomic information about all types of anaerobically, and then activity assays for the reduction of 5-HMF were organisms has proven to be very useful for the identification of NAD(P) performed [180]. In another study, Rellos et al. generated a library of H-dependent oxidoreductases, increasing the number of members of variants of Zymomonas mobilis alcohol dehydrogenase 2, an NADH-de- known types and thereby improving understanding of conserved fea- pendent, iron-activated alcohol dehydrogenase able to oxidize only tures, but also leading to identification of new types such as the ATP ethanol, 1-propanol and allyl alcohol. The library of variants was and NADPH-dependent CAR activity described in section 3, EC 1.2.1. In generated by a combination of error-prone PCR and site-directed mu- parallel, the acceleration in the rate at which protein structures are tagenesis, and transformants were screened by a colorimetric assay determined and made public is providing more insight into the struc- with butanol. Variants with higher activity towards butanol than to tural basis of aspects such as cofactor and substrate specificity, in- ethanol, as well as variants able to use NADPH/NADP+ while still re- formation which is important to guide protein engineering. Further- taining the ability to use NADH/NAD+, were identified. Additionally, more, the ability to exploit this increasing knowledge base has been another variant was able to use Zn instead of Fe as the metallic cofactor facilitated by the development of protein engineering and directed [181]. There have also been several cases where the enantiospecificity evolution approaches which allow the properties of enzymes to be of an alcohol dehydrogenase has been altered. However, most fre- tailored in rational and semi-rational ways. There has been practical quently the reversal of enantiospecificity is not total, comes at the cost progress in the prediction of enzyme function from structure: The new of decreased catalytic efficiency or produces unexpected side-effects. CSR-SALAD computational tool uses recently-established rules to in- For example, Akita et al. generated a thermostable NADPH-dependent terpret structural and sequence information in order to predict specific D-amino acid dehydrogenase (DAADH) from Ureibacillus thermo- mutations which might allow reversal of the cofactor specificity of a sphaericus meso-diaminopimelate dehydrogenase (DAPDH) by in- given NAD(P)H-dependent oxidoreductase [66]. The rapid growth in troducing five point mutations in the active site that had been pre- sequence and structural information together with technological de- viously described to be responsible for converting another DAPDH from velopments will undoubtedly expand still further the potential and a mesophilic organism (Corynebacterium glutamicum) into a DAADH usefulness of an already naturally versatile group of enzymes. [182]. Interestingly, while the resulting variant was able to oxidize several D-amino acids, it was no longer able to oxidize meso-diamino- Transparency document pimelate [183]. Finally, another quality usually sought during the screening of Supplementary data to this article can be found online at https:// variant libraries is increased thermal stability. Increasing thermal sta- doi.org/10.1016/j.bbapap.2017.11.005. bility is not only desirable for achieving a reduced loss of catalyst during enzyme-catalyzed reactions, but also because the catalyzed re- Transparency document action takes place at a higher rate when temperature is increased if the enzyme can withstand the higher temperature. For example, a dramatic The Transparency document associated with this article can be increase in the thermal stability of phosphite dehydrogenase was found in the online version. achieved by Johannes et al., who identified a mutant with a T50 (re- quired temperature to reduce the initial enzyme activity by 50%) Acknowledgments higher than the wild type by 20 °C and a 7000-fold greater half-life at 45 °C, which could be used as an efficient way to regenerate reduced The authors gratefully acknowledge funding from the Biotechnology NAD(P)H in industrial biocatalysis applications. In this case, the ap- and Biological Sciences Research Council (BBSRC) and Syngenta for proach involved a combination of error-prone PCR and site-directed CASE studentship BB/N503873/1, and the BBSRC for grants BB/ mutagenesis. Three rounds of error-prone PCR were carried out, M011321/1 and BB/M002454/1. starting with the wild type gene. After each round of error-prone PCR, variants with improved thermal stability were identified. The best References variants were sequenced, and the mutations found in all variants were incorporated into the template. The new template incorporating all the [1] J.D. Stewart, Future directions in alcohol dehydrogenase-catalyzed reactions, – mutations served as the template for the next round of error-prone PCR Future Directions in Biocatalysis, Elsevier, 2007, pp. 293 304. [2] R. Meijers, R.J. Morris, H.W. Adolph, A. Merli, V.S. Lamzin, E.S. Cedergren- [184]. Zeppezauer, On the enzymatic activation of NADH, J. Biol. Chem. 276 (2001) Many attempts at modifying the substrate and cofactor specificity of 9316–9321. [3] A.K. Holm, L.M. Blank, M. Oldiges, A. Schmid, C. Solem, P.R. Jensen, G.N. Vemuri, NAD(P)H-dependent oxidoreductases have been carried out and these Metabolic and transcriptional response to cofactor perturbations in Escherichia coli, attempts have been successful to a certain extent. However, in many J. Biol. Chem. 285 (2010) 17498–17506. cases the mutations do not achieve the desired effect, cause the loss of [4] U. Sauer, F. Canonaco, S. Heri, A. Perrenoud, E. Fischer, The soluble and mem- brane-bound transhydrogenases UdhA and PntAB have divergent functions in catalytic activity or lead to unexpected results [185,186]. This is par- NADPH metabolism of Escherichia coli, J. Biol. Chem. 279 (2004) 6613–6619. tially due to the lack of understanding of the relationship between the [5] M.R. de Graef, S. Alexeeva, J.L. Snoep, M.J. Teixeira de Mattos, The steady-state fl amino acid sequence of a protein, its structure and its function, which internal redox state (NADH/NAD) re ects the external redox state and is corre- lated with catabolic adaptation in Escherichia coli, J. Bacteriol. 181 (1999) still prevents us from developing widely applicable methodologies to 2351–2357. alter the substrate or cofactor specificity of oxidoreductases. [6] C. Auriol, G. Bestel-Corre, J.-B. Claude, P. Soucaille, I. Meynial-Salles, Stress-in- duced evolution of Escherichia coli points to original concepts in respiratory co- factor selectivity, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 1278–1283. 6. Conclusion and future perspective [7] T. Fuhrer, U. Sauer, Different biochemical mechanisms ensure network-wide balancing of reducing equivalents in microbial metabolism, J. Bacteriol. 191 (2009) 2112–2121. NAD(P)H-dependent oxidoreductases constitute an extremely di- [8] L. Hue, The role of futile cycles in the regulation of carbohydrate metabolism in verse group of enzymes able to act on a similarly vast range of the liver, Adv. Enzymol. Relat. Areas Mol. Biol. 52 (1981) 247–331.

344 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

[9] S.W. May, Applications of oxidoreductases, Curr. Opin. Biotechnol. 10 (1999) reductase for iron uptake from soils, Nature 397 (1999) 694–697. 370–375. [43] T. Nishino, K. Okamoto, Mechanistic insights into xanthine oxidoreductase from [10] C.M. Nealon, M.M. Musa, J.M. Patel, R.S. Phillips, Controlling substrate specificity development studies of candidate drugs to treat hyperuricemia and gout, J. Biol. and stereospecificity of alcohol dehydrogenases, ACS Catal. 5 (2015) 2100–2114. Inorg. Chem. 20 (2015) 195–207. [11] W. Hummel, Large-scale applications of NAD(P)-dependent oxidoreductases: re- [44] C. Maugé, T. Granier, B.L. d'Estaintot, M. Gargouri, C. Manigand, J.-M. Schmitter, cent developments, Trends Biotechnol. 17 (1999) 487–492. J. Chaudière, B. Gallois, Crystal structure and catalytic mechanism of leu- [12] Y.-G. Zheng, H.-H. Yin, D.-F. Yu, X. Chen, X.-L. Tang, X.-J. Zhang, Y.-P. Xue, Y.- coanthocyanidin reductase from Vitis vinifera, J. Mol. Biol. 397 (2010) 1079–1091. J. Wang, Z.-Q. Liu, Recent advances in biotechnological applications of alcohol [45] N. Carrillo, E.A. Ceccarelli, Open questions in ferredoxin-NADP+ reductase cat- dehydrogenases, Appl. Microbiol. Biotechnol. 101 (2017) 987–1001. alytic mechanism, Eur. J. Biochem. 270 (2003) 1900–1915. [13] B.R. Lichtenstein, T.A. Farid, G. Kodali, L.A. Solomon, J.L.R. Anderson, [46] K. Ma, M.W. Adams, A hyperactive NAD(P)H:Rubredoxin oxidoreductase from the M.M. Sheehan, N.M. Ennist, B.A. Fry, S.E. Chobot, C. Bialas, J.A. Mancini, hyperthermophilic archaeon Pyrococcus furiosus, J. Bacteriol. 181 (1999) C.T. Armstrong, Z. Zhao, T.V. Esipova, D. Snell, S.A. Vinogradov, B.M. Discher, 5530–5533. C.C. Moser, P.L. Dutton, Engineering oxidoreductases: maquette proteins designed [47] I.F. Sevrioukova, H. Li, T.L. Poulos, Crystal structure of putidaredoxin reductase from scratch, Biochem. Soc. Trans. 40 (2012) 561–566. from Pseudomonas putida, the final structural component of the cytochrome [14] F. Xu, Applications of oxidoreductases: recent progress, Ind. Biotechnol. 1 (2005) P450cam monooxygenase, J. Mol. Biol. 336 (2004) 889–902. 38–50. [48] H. Sakamoto, M. Ohta, R. Miura, T. Sugiyama, T. Yamano, Y. Miyake, Studies on [15] K.L. Kavanagh, H. Jörnvall, B. Persson, U. Oppermann, Medium- and short-chain the reaction mechanism of NADPH-adrenodoxin reductase with NADPH, J. dehydrogenase/reductase gene and protein families: the SDR superfamily: func- Biochem. 92 (1982) 1941–1950. tional and structural diversity within a family of metabolic and regulatory en- [49] C.M. Jenkins, M.R. Waterman, NADPH-flavodoxin reductase and flavodoxin from zymes, Cell. Mol. Life Sci. 65 (2008) 3895–3906. Escherichia coli: characteristics as a soluble microsomal P450 reductase, [16] O. de Smidt, J.C. du Preez, J. Albertyn, The alcohol dehydrogenases of Biochemistry 37 (1998) 6106–6113. Saccharomyces cerevisiae: a comprehensive review, FEMS Yeast Res. 8 (2008) [50] H.A. Relyea, W.A. van der Donk, Mechanism and applications of phosphite de- 967–978. hydrogenase, Bioorg. Chem. 33 (2005) 171–189. [17] V. Koppaka, D.C. Thompson, Y. Chen, M. Ellermann, K.C. Nicolaou, R.O. Juvonen, [51] J. Hu, W. Chuenchor, S.E. Rokita, A switch between one- and two-electron D. Petersen, R.A. Deitrich, T.D. Hurley, V. Vasiliou, Aldehyde dehydrogenase in- chemistry of the human flavoprotein iodotyrosine deiodinase is controlled by hibitors: a comprehensive review of the pharmacology, mechanism of action, substrate, J. Biol. Chem. 290 (2015) 590–600. substrate specificity, and clinical application, Pharmacol. Rev. 64 (2012) 520–539. [52] K.A. Payne, C.P. Quezada, K. Fisher, M.S. Dunstan, F.A. Collins, H. Sjuts, C. Levy, [18] K. Napora-Wijata, G.A. Strohmeier, M. Winkler, Biocatalytic reduction of car- S. Hay, S.E. Rigby, D. Leys, Reductive dehalogenase structure suggests a me- boxylic acids, Biotechnol. J. 9 (2014) 822–843. chanism for B12-dependent dehalogenation, Nature 517 (2015) 513–516. [19] H.S. Toogood, N.S. Scrutton, New developments in “ene”-reductase catalysed [53] T. Min, H. Kasahara, D.L. Bedgar, B. Youn, P.K. Lawrence, D.R. Gang, S.C. Halls, biological hydrogenations, Curr. Opin. Chem. Biol. 19 (2014) 107–115. H. Park, J.L. Hilsenbeck, L.B. Davin, N.G. Lewis, C. Kang, Crystal structures of [20] N.M. Brunhuber, J.S. Blanchard, The biochemistry and enzymology of amino acid pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their dehydrogenases, Crit. Rev. Biochem. Mol. Biol. 29 (1994) 415–467. relationship to isoflavone reductases, J. Biol. Chem. 278 (2003) 50714–50723. [21] K.I. Varughese, N.H. Xuong, P.M. Kiefer, D.A. Matthews, J.M. Whiteley, Structural [54] R.D. Gupta, Recent advances in , Sustain. Chem. Process. 4 and mechanistic characteristics of dihydropteridine reductase: a member of the (2016) 2. Tyr-(Xaa)3-Lys-containing family of reductases and dehydrogenases, Proc. Natl. [55] B. Arora, J. Mukherjee, M.N. Gupta, Enzyme promiscuity: using the dark side of Acad. Sci. U. S. A. 91 (1994) 5582–5586. enzyme specificity in white biotechnology, Sustain. Chem. Process. 2 (2014) 25. [22] J.R. Schnell, H.J. Dyson, P.E. Wright, Structure, dynamics, and catalytic function [56] B. Ferreira-Silva, I. Lavandera, A. Kern, K. Faber, W. Kroutil, Chemo-promiscuity of dihydrofolate reductase, Annu. Rev. Biophys. Biomol. Struct. 33 (2004) of alcohol dehydrogenases: reduction of phenylacetaldoxime to the alcohol, 119–140. Tetrahedron 66 (2010) 3410–3414. [23] S.C. Tu, Reduced flavin: donor and acceptor enzymes and mechanisms of chan- [57] P.J. O'Brien, D. Herschlag, Catalytic promiscuity and the evolution of new enzy- neling, Antioxid. Redox Signal. 3 (2001) 881–897. matic activities, Chem. Biol. 6 (1999) R91–R105. [24] A. Pedersen, G.B. Karlsson, J. Rydström, Proton-translocating transhydrogenase: [58] C. Lee, H. Görisch, H. Kleinkauf, R. Zocher, A highly specific D-hydroxyisovalerate an update of unsolved and controversial issues, J. Bioenerg. Biomembr. 40 (2008) dehydrogenase from the enniatin producer Fusarium sambucinum, J. Biol. Chem. 463–473. 267 (1992) 11741–11744. [25] G. Voordouw, S.M. van der Vies, A.P. Themmen, Why are two different types of [59] V. Höllrigl, F. Hollmann, A.C. Kleeb, K. Buehler, A. Schmid, TADH, the thermo- pyridine nucleotide transhydrogenase found in living organisms? Eur. J. Biochem. stable alcohol dehydrogenase from Thermus sp. ATN1: a versatile new biocatalyst 131 (1983). for organic synthesis, Appl. Microbiol. Biotechnol. 81 (2008) 263–273. [26] C. Sparacino-Watkins, J.F. Stolz, P. Basu, Nitrate and periplasmic nitrate re- [60] S. Kinoshita, T. Kakizono, K. Kadota, K. Das, H. Taguchi, Purification of two al- ductases, Chem. Soc. Rev. 43 (2014) 676–706. cohol dehydrogenases from Zymomonas mobilis and their properties, Appl. [27] N. Castiglione, S. Rinaldo, G. Giardina, V. Stelitano, F. Cutruzzolà, Nitrite and Microbiol. Biotechnol. 22 (1985) 249–254. nitrite reductases: from molecular mechanisms to significance in human health [61] F. Kaufmann, D.R. Lovley, Isolation and characterization of a soluble NADPH- and disease, Antioxid. Redox Signal. 17 (2012) 684–716. dependent Fe(III) reductase from Geobacter sulfurreducens, J. Bacteriol. 183 (2001) [28] M.D. Roldán, E. Pérez-Reinado, F. Castillo, C. Moreno-Vivián, Reduction of poly- 4468–4476. nitroaromatic compounds: the bacterial nitroreductases, FEMS Microbiol. Rev. 32 [62] E. Di Luccio, R.A. Elling, D.K. Wilson, Identification of a novel NADH-specific (2008) 474–500. aldo-keto reductase using sequence and structural homologies, Biochem. J. 400 [29] B.R. Crane, L.M. Siegel, E.D. Getzoff, Probing the catalytic mechanism of sulfite (2006) 105–114. reductase by X-ray crystallography: structures of the Escherichia coli hemoprotein [63] C.N. Jensen, J. Cartwright, J. Ward, S. Hart, J.P. Turkenburg, S.T. Ali, M.J. Allen, in complex with substrates, inhibitors, intermediates, and products, Biochemistry G. Grogan, A flavoprotein monooxygenase that catalyses a Baeyer–Villiger reac- 36 (1997) 12120–12137. tion and thioether oxidation using NADH as the nicotinamide cofactor, [30] N. Couto, J. Wood, J. Barber, The role of glutathione reductase and related en- Chembiochem 13 (2012) 872–878. zymes on cellular redox homoeostasis network, Free Radic. Biol. Med. 95 (2016) [64] R. Woodyer, M. Simurdiak, W.A. van der Donk, H. Zhao, Heterologous expression, 27–42. purification, and characterization of a highly active xylose reductase from [31] D. Mustacich, G. Powis, Thioredoxin reductase, Biochem. J. 346 (Pt 1) (2000) 1–8. Neurospora crassa, Appl. Environ. Microbiol. 71 (2005) 1642–1647. [32] R.P. Hopkins, E.C. Drummond, P. Callaghan, Dehydrogenation of trans-ace- [65] K. Sueyoshi, A. Kleinhofs, R.L. Warner, Expression of NADH-specific and NAD(P) naphthene-1,2-diol by liver cytosol preparations, Biochem. Soc. Trans. 1 (1973) H-bispecific nitrate reductase genes in response to nitrate in barley, Plant Physiol. 989–991. 107 (1995) 1303–1311. [33] T. Stehle, A. Claiborne, G.E. Schulz, NADH binding site and catalysis of NADH [66] J.K.B. Cahn, C.A. Werlang, A. Baumschlager, S. Brinkmann-Chen, S.L. Mayo, peroxidase, Eur. J. Biochem. 211 (1993) 221–226. F.H. Arnold, A general tool for engineering the NAD/NADP cofactor preference of [34] W. Lubitz, H. Ogata, O. Rüdiger, E. Reijerse, Hydrogenases, Chem. Rev. 114 oxidoreductases, ACS Synth. Biol. (2016), http://dx.doi.org/10.1021/acssynbio. (2014) 4081–4148. 6b00188. [35] A.M. McDonnell, C.H. Dang, Basic review of the cytochrome p450 system, J. Adv. [67] S. Dey, Z. Hu, X.L. Xu, J.C. Sacchettini, G.A. Grant, The effect of hinge mutations Pract. Oncol. 4 (2013) 263–268. on effector binding and domain rotation in Escherichia coli D-3-phosphoglycerate [36] M. Palrasu, S. Nagini, Cytochrome P450 structure, function and clinical sig- dehydrogenase, J. Biol. Chem. 282 (2007) 18418–18426. nificance: a review, Curr. Drug Targets 18 (2017), https://www.ncbi.nlm.nih.gov/ [68] J.A. Read, V.J. Winter, C.M. Eszes, R.B. Sessions, R.L. Brady, Structural basis for /28124606. altered activity of M- and H-isozyme forms of human lactate dehydrogenase, [37] M.M.E. Huijbers, S. Montersino, A.H. Westphal, D. Tischler, W.J.H. van Berkel, Proteins 43 (2001) 175–185. Flavin dependent monooxygenases, Arch. Biochem. Biophys. 544 (2014) 2–17. [69] M.J. Adams, G.C. Ford, R. Koekoek, P.J. Lentz, A. McPherson Jr, M.G. Rossmann, [38] V.C.-C. Wang, S. Maji, P.P.-Y. Chen, H.K. Lee, S.S.-F. Yu, S.I. Chan, Alkane oxi- I.E. Smiley, R.W. Schevitz, A.J. Wonacott, Structure of lactate dehydrogenase at dation: methane monooxygenases, related enzymes, and their biomimetics, Chem. 2–8 A resolution, Nature 227 (1970) 1098–1103. Rev. 117 (2017) 8574–8621. [70] M.G. Rossmann, D. Moras, K.W. Olsen, Chemical and biological evolution of nu- [39] T. Barkay, S.M. Miller, A.O. Summers, Bacterial mercury resistance from atoms to cleotide-binding protein, Nature 250 (1974) 194–199. ecosystems, FEMS Microbiol. Rev. 27 (2003) 355–384. [71] G.E. Schulz, R.H. Schirmer, E.F. Pai, FAD-binding site of glutathione reductase, J. [40] S. Cheng, T.A. Bobik, Characterization of the PduS cobalamin reductase of sal- Mol. Biol. 160 (1982) 287–308. monella enterica and its role in the Pdu microcompartment, J. Bacteriol. 192 [72] I. Hanukoglu, T. Gutfinger, cDNA sequence of adrenodoxin reductase. (2010) 5071–5080. Identification of NADP-binding sites in oxidoreductases, Eur. J. Biochem. 180 [41] F. Watanabe, R. Yamaji, Y. Isegawa, T. Yamamoto, Y. Tamura, Y. Nakano, (1989) 479–484. Characterization of aquacobalamin reductase (NADPH) from Euglena gracilis, Arch. [73] C.A. Bottoms, P.E. Smith, J.J. Tanner, A structurally conserved water molecule in Biochem. Biophys. 305 (1993) 421–427. Rossmann dinucleotide-binding domains, Protein Sci. 11 (2002) 2125–2137. [42] N.J. Robinson, C.M. Procter, E.L. Connolly, M.L. Guerinot, A ferric-chelate [74] I. Hanukoglu, Proteopedia: Rossmann fold: A beta-alpha-beta fold at dinucleotide

345 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

binding sites, Biochem. Mol. Biol. Educ. 43 (2015) 206–209. Escherichia coli, Metab. Eng. 22 (2014) 10–21. [75] A.S. Kutzenko, V.S. Lamzin, V.O. Popov, Conserved supersecondary structural [104] C.K. Winkler, G. Tasnádi, D. Clay, M. Hall, K. Faber, Asymmetric bioreduction of motif in NAD-dependent dehydrogenases, FEBS Lett. 423 (1998) 105–109. activated alkenes to industrially relevant optically active compounds, J. [76] K.L. Kavanagh, H. Jörnvall, B. Persson, U. Oppermann, Medium-and short-chain Biotechnol. 162 (2012) 381–389. dehydrogenase/reductase gene and protein families, Cell. Mol. Life Sci. 65 (2008) [105] O. Warburg, W. Christian, Über das gelbe Ferment und seine Wirkungen, Biochem. 3895–3906. Z. 266 (1933) 377–411. [77] M. Bashton, C. Chothia, The geometry of domain combination in proteins, J. Mol. [106] R.E. Williams, N.C. Bruce, “New uses for an old enzyme”—the old yellow enzyme Biol. 315 (2002) 927–939. family of flavoenzymes, Microbiology 148 (2002) 1607–1614. [78] P.A. Frey, A.D. Hegeman, Enzymatic Reaction Mechanisms, Oxford University [107] H. Theorell, Preparation in pure state of the effect group of yellow enzymes, Press, 2007. Biochem. Z. 275 (1935) 344–346. [79] P.J. Baker, K.L. Britton, M. Fisher, J. Esclapez, C. Pire, M.J. Bonete, J. Ferrer, [108] H. Theorell, Nobel Prize Lecture, (1955). D.W. Rice, Active site dynamics in the zinc-dependent medium chain alcohol [109] B. Boonstra, D.A. Rathbone, N.C. Bruce, Engineering novel biocatalytic routes for dehydrogenase superfamily, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 779–784. production of semisynthetic opiate drugs, Biomol. Eng. 18 (2001) 41–47. [80] K. Ma, M.W. Adams, An unusual oxygen-sensitive, iron- and zinc-containing al- [110] N.M. Brunhuber, J.B. Thoden, J.S. Blanchard, J.L. Vanhooke, Rhodococcus L-phe- cohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus,J. nylalanine dehydrogenase: kinetics, mechanism, and structural basis for catalytic Bacteriol. 181 (1999) 1163–1170. specificity, Biochemistry 39 (2000) 9174–9187. [81] X. Liu, Y. Dong, J. Zhang, A. Zhang, L. Wang, L. Feng, Two novel metal-in- [111] T.J. Stillman, P.J. Baker, K.L. Britton, D.W. Rice, Conformational flexibility in dependent long-chain alkyl alcohol dehydrogenases from Geobacillus thermo- glutamate dehydrogenase. Role of water in substrate recognition and catalysis, J. denitrificans NG80-2, Microbiology 155 (2009) 2078–2085. Mol. Biol. 234 (1993) 1131–1139. [82] K.L. Kavanagh, M. Klimacek, B. Nidetzky, D.K. Wilson, Crystal structure of [112] P.J. Baker, K.L. Britton, P.C. Engel, G.W. Farrants, K.S. Lilley, D.W. Rice, Pseudomonas fluorescens mannitol 2-dehydrogenase binary and ternary complexes T.J. Stillman, Subunit assembly and active site location in the structure of gluta- specificity and catalytic mechanism, J. Biol. Chem. 277 (2002) 43433–43442. mate dehydrogenase, Proteins 12 (1992) 75–86. [83] C. Gaona-López, A. Julián-Sánchez, H. Riveros-Rosas, Diversity and evolutionary [113] P.J. Baker, M.L. Waugh, X.G. Wang, T.J. Stillman, A.P. Turnbull, P.C. Engel, analysis of iron-containing (type-III) alcohol dehydrogenases in eukaryotes, PLoS D.W. Rice, Determinants of substrate specificity in the superfamily of amino acid One 11 (2016) e0166851. dehydrogenases, Biochemistry 36 (1997) 16109–16115. [84] C. Montella, L. Bellsolell, R. Pérez-Luque, J. Badía, L. Baldoma, M. Coll, J. Aguilar, [114] Q. Wan, B.C. Bennett, M.A. Wilson, A. Kovalevsky, P. Langan, E.E. Howell, Crystal structure of an iron-dependent group III dehydrogenase that interconverts C. Dealwis, Toward resolving the catalytic mechanism of dihydrofolate reductase L-lactaldehyde and L-1,2-propanediol in Escherichia coli, J. Bacteriol. 187 (2005) using neutron and ultrahigh-resolution X-ray crystallography, Proc. Natl. Acad. 4957–4966. Sci. U. S. A. 111 (2014) 18225–18230. [85] J.-H. Moon, H.-J. Lee, S.-Y. Park, J.-M. Song, M.-Y. Park, H.-M. Park, J. Sun, J.- [115] C.T. Liu, K. Francis, J.P. Layfield, X. Huang, S. Hammes-Schiffer, A. Kohen, H. Park, B.Y. Kim, J.-S. Kim, Structures of iron-dependent alcohol dehydrogenase S.J. Benkovic, Escherichia coli dihydrofolate reductase catalyzed proton and hy- 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor, J. Mol. Biol. dride transfers: temporal order and the roles of Asp27 and Tyr100, Proc. Natl. 407 (2011) 413–424. Acad. Sci. U. S. A. 111 (2014) 18231–18236. [86] E. Cabiscol, J. Aguilar, J. Ros, Metal-catalyzed oxidation of Fe2+ dehydrogenases. [116] S.F. Queener, V. Cody, J. Pace, P. Torkelson, A. Gangjee, Trimethoprim resistance Consensus target sequence between propanediol oxidoreductase of Escherichia of dihydrofolate reductase variants from clinical isolates of Pneumocystis jirovecii, coli and alcohol dehydrogenase II of Zymomonas mobilis, J. Biol. Chem. 269 (9) Antimicrob. Agents Chemother. 57 (2013) 4990–4998. (1994) 6592–6597 (ASBMB. (1994)), http://www.jbc.org/content/269/9/6592. [117] G. Voordouw, S.M. van der Vies, A.P. Themmen, Why are two different types of short. pyridine nucleotide transhydrogenase found in living organisms? Eur. J. Biochem. [87] J. Tamarit, E. Cabiscol, J. Aguilar, J. Ros, Differential inactivation of alcohol de- 131 (1983) 527–533. hydrogenase isoenzymes in Zymomonas mobilis by oxygen, J. Bacteriol. 179 (1997) [118] J.B. Hoek, J. Rydström, Physiological roles of nicotinamide nucleotide transhy- 1102–1104. drogenase, Biochem. J. 254 (1988) 1–10. [88] L. Zhang, B. Ahvazi, R. Szittner, A. Vrielink, E. Meighen, A histidine residue in the [119] A.M. Sanchez, J. Andrews, I. Hussein, G.N. Bennett, K.-Y. San, Effect of over- catalytic mechanism distinguishes Vibrio harveyi aldehyde dehydrogenase from expression of a soluble pyridine nucleotide transhydrogenase (UdhA) on the other members of the aldehyde dehydrogenase superfamily, Biochemistry 39 production of poly(3-hydroxybutyrate) in Escherichia coli, Biotechnol. Prog. 22 (2000) 14409–14418. (2006) 420–425. [89] J. Hempel, I. Kuo, J. Perozich, B.C. Wang, R. Lindahl, H. Nicholas, Aldehyde de- [120] H. Niederholtmeyer, B.T. Wolfstädter, D.F. Savage, P.A. Silver, J.C. Way, hydrogenase. Maintaining critical active site geometry at motif 8 in the class 3 Engineering cyanobacteria to synthesize and export hydrophilic products, Appl. enzyme, Eur. J. Biochem. 268 (2001) 722–726. Environ. Microbiol. 76 (2010) 3462–3466. [90] S.A. Marchitti, C. Brocker, D. Stagos, V. Vasiliou, Non-P450 aldehyde oxidizing [121] A. Weckbecker, W. Hummel, Improved synthesis of chiral alcohols with Escherichia enzymes: the aldehyde dehydrogenase superfamily, Expert Opin. Drug Metab. coli cells co-expressing pyridine nucleotide transhydrogenase, NADP+-dependent Toxicol. 4 (2008) 697–720. alcohol dehydrogenase and NAD+-dependent formate dehydrogenase, Biotechnol. [91] D.P. Clark, The fermentation pathways of Escherichia coli, FEMS Microbiol. Rev. 5 Lett. 26 (2004) 1739–1744. (1989) 223–234. [122] J. Jan, I. Martinez, Y. Wang, G.N. Bennett, K.-Y. San, Metabolic engineering and [92] R. Gheshlaghi, J.M. Scharer, M. Moo-Young, C.P. Chou, Metabolic pathways of transhydrogenase effects on NADPH availability in Escherichia coli, Biotechnol. clostridia for producing butanol, Biotechnol. Adv. 27 (2009) 764–781. Prog. 29 (2013) 1124–1130. [93] H. Seedorf, W.F. Fricke, B. Veith, H. Brüggemann, H. Liesegang, A. Strittmatter, [123] M. AbuKhader, J. Heap, C. De Matteis, B. Kellam, S.W. Doughty, N. Minton, M. Miethke, W. Buckel, J. Hinderberger, F. Li, C. Hagemeier, R.K. Thauer, M. Paoli, Binding of the anticancer prodrug CB1954 to the activating enzyme G. Gottschalk, The genome of Clostridium kluyveri, a strict anaerobe with unique NQO2 revealed by the crystal structure of their complex, J. Med. Chem. 48 (2005) metabolic features, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 2128–2133. 7714–7719. [94] Y. Dekishima, E.I. Lan, C.R. Shen, K.M. Cho, J.C. Liao, Extending carbon chain [124] C. Moreno-Vivián, P. Cabello, M. Martínez-Luque, R. Blasco, F. Castillo, length of 1-butanol pathway for 1-hexanol synthesis from glucose by engineered Prokaryotic nitrate reduction: molecular properties and functional distinction Escherichia coli, J. Am. Chem. Soc. 133 (2011) 11399–11401. among bacterial nitrate reductases, J. Bacteriol. 181 (1999) 6573–6584. [95] C. Dellomonaco, J.M. Clomburg, E.N. Miller, R. Gonzalez, Engineered reversal of [125] K. Fischer, G.G. Barbier, H.-J. Hecht, R.R. Mendel, W.H. Campbell, G. Schwarz, the β-oxidation cycle for the synthesis of fuels and chemicals, Nature 476 (2011) Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate 355–359. reductase active site, Plant Cell 17 (2005) 1167–1179. [96] R.T. Yan, J.S. Chen, Coenzyme A-acylating aldehyde dehydrogenase from [126] P.A. Janick, D.C. Rueger, R.J. Krueger, M.J. Barber, L.M. Siegel, Characterization Clostridium beijerinckii NRRL B592, Appl. Environ. Microbiol. 56 (1990) of complexes between Escherichia coli sulfite reductase hemoprotein subunit and 2591–2599. its substrates sulfite and nitrite, Biochemistry 22 (1983) 396–408. [97] J. Membrillo-Hernández, P. Echave, E. Cabiscol, J. Tamarit, J. Ros, E.C.C. Lin, [127] L.M. Siegel, P.S. Davis, Reduced nicotinamide adenine dinucleotide phosphate- Evolution of the adhE gene product of Escherichia coli from a functional reductase sulfite reductase of enterobacteria. IV. The Escherichia coli hemoflavoprotein: to a dehydrogenase: genetic and biochemical studies of the mutant proteins, J. subunit structure and dissociation into hemoprotein and flavoprotein components, Biol. Chem. 275 (2000) 33869–33875. J. Biol. Chem. 249 (1974) 1587–1598. [98] J. Lo, T. Zheng, S. Hon, D.G. Olson, L.R. Lynd, The bifunctional alcohol and al- [128] A. Gaber, M. Tamoi, T. Takeda, Y. Nakano, S. Shigeoka, NADPH-dependent glu- dehyde dehydrogenase gene, adhE, is necessary for ethanol production in tathione peroxidase-like proteins (Gpx-1, Gpx-2) reduce unsaturated fatty acid Clostridium thermocellum and Thermoanaerobacterium saccharolyticum, J. Bacteriol. hydroperoxides in Synechocystis PCC 6803, FEBS Lett. 499 (2001) 32–36. 197 (2015) 1386–1393. [129] A. Volbeda, M.H. Charon, C. Piras, E.C. Hatchikian, Crystal structure of the [99] c. D. Cloning, E. Relationships, T. Distribution, CoA-dependent methylmalonate- nickel–iron hydrogenase from Desulfovibrio gigas, http://www.nature.com/nature/ semialdehyde dehydrogenase, a unique member of the aldehyde dehydrogenase journal/v373/n6515/abs/373580a0.html, (1995). superfamily, Plan. Perspect. 19724 (1992) 19729. [130] A. Volbeda, E. Garcin, C. Piras, Structure of the hydrogenase active site: evidence [100] M.K. Akhtar, N.J. Turner, P.R. Jones, Carboxylic acid reductase is a versatile en- for biologically uncommon Fe ligands, J. Amer. Chem. Soc. 7863 (1996) zyme for the conversion of fatty acids into fuels and chemical commodities, Proc. 12989–12996. Natl. Acad. Sci. U. S. A. 110 (2013) 87–92. [131] C. Fichtner, C. Laurich, E. Bothe, W. Lubitz, Spectroelectrochemical character- [101] W. Wang, H. Wei, E. Knoshaug, S. Van Wychen, Q. Xu, M.E. Himmel, M. Zhang, ization of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, Fatty alcohol production in Lipomyces starkeyi and Yarrowia lipolytica, Biotechnol. Biochemistry 45 (2006) 9706–9716. Biofuels. 9 (2016) 227. [132] A. Volbeda, L. Martin, C. Cavazza, M. Matho, B.W. Faber, W. Roseboom, [102] L. Crépin, E. Lombard, S.E. Guillouet, Metabolic engineering of Cupriavidus necator S.P.J. Albracht, E. Garcin, M. Rousset, J.C. Fontecilla-Camps, Structural differ- for heterotrophic and autotrophic alka(e)ne production, Metab. Eng. 37 (2016) ences between the ready and unready oxidized states of [NiFe] hydrogenases, J. 92–101. Biol. Inorg. Chem. 10 (2005) 239–249. [103] R. Liu, F. Zhu, L. Lu, A. Fu, J. Lu, Z. Deng, T. Liu, Metabolic engineering of fatty [133] J.W. Peters, X-ray crystal structure of the Fe-only hydrogenase (CpI) from acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Clostridium pasteurianum to 1.8 Angstrom resolution, Science 282 (1998)

346 L. Sellés Vidal et al. BBA - Proteins and Proteomics 1866 (2018) 327–347

1853–1858. [161] S. Atsumi, A.F. Cann, M.R. Connor, C.R. Shen, K.M. Smith, M.P. Brynildsen, [134] E.M. Shepard, B.R. Duffus, S.J. George, S.E. McGlynn, M.R. Challand, K.J.Y. Chou, T. Hanai, J.C. Liao, Metabolic engineering of Escherichia coli for 1- K.D. Swanson, P.L. Roach, S.P. Cramer, J.W. Peters, J.B. Broderick, [FeFe]-hy- butanol production, Metab. Eng. 10 (2008) 305–311. drogenase maturation: HydG-catalyzed synthesis of carbon monoxide, J. Am. [162] O.V. Berezina, N.V. Zakharova, A. Brandt, S.V. Yarotsky, W.H. Schwarz, Chem. Soc. 132 (2010) 9247–9249. V.V. Zverlov, Reconstructing the clostridial n-butanol metabolic pathway in [135] P.M. Vignais, B. Billoud, Occurrence, classification, and biological function of Lactobacillus brevis, Appl. Microbiol. Biotechnol. 87 (2010) 635–646. hydrogenases: an overview, Chem. Rev. 107 (2007) 4206–4272. [163] J.W.K. Oliver, I.M.P. Machado, H. Yoneda, S. Atsumi, Cyanobacterial conversion [136] T. Burgdorf, E. van der Linden, M. Bernhard, Q.Y. Yin, J.W. Back, A.F. Hartog, of carbon dioxide to 2,3-butanediol, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) A.O. Muijsers, C.G. de Koster, S.P.J. Albracht, B. Friedrich, The soluble NAD+- 1249–1254. reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits [164] S.-J. Ha, J.M. Galazka, S.R. Kim, J.-H. Choi, X. Yang, J.-H. Seo, N.L. Glass, and can be specifically activated by NADPH, J. Bacteriol. 187 (2005) 3122–3132. J.H.D. Cate, Y.-S. Jin, Engineered Saccharomyces cerevisiae capable of simultaneous [137] D.E. Torres Pazmiño, M. Winkler, A. Glieder, M.W. Fraaije, Monooxygenases as cellobiose and xylose fermentation, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) biocatalysts: classification, mechanistic aspects and biotechnological applications, 504–509. J. Biotechnol. 146 (2010) 9–24. [165] J.Y. Lee, J.R. Roh, H.S. Kim, Metabolic engineering of Pseudomonas putida for the [138] A. Dey, Y. Jiang, P. Ortiz de Montellano, K.O. Hodgson, B. Hedman, E.I. Solomon, simultaneous biodegradation of benzene, toluene, and p-xylene mixture, S K-edge XAS and DFT calculations on cytochrome P450: covalent and ionic Biotechnol. Bioeng. 43 (1994) 1146–1152. contributions to the cysteine-Fe bond and their contribution to reactivity, J. Am. [166] S.D. Finley, L.J. Broadbelt, V. Hatzimanikatis, In silico feasibility of novel biode- Chem. Soc. 131 (2009) 7869–7878. gradation pathways for 1,2,4-trichlorobenzene, BMC Syst. Biol. 4 (2010) 7. [139] E.T. Farinas, U. Schwaneberg, A. Glieder, F.H. Arnold, Directed evolution of a [167] H. Li, J. Chen, Y. Li, Enhanced activity of yqhD oxidoreductase in synthesis of 1,3- cytochrome P450 monooxygenase for alkane oxidation, Adv. Synth. Catal. 343 propanediol by error-prone PCR, Prog. Nat. Sci. 18 (2008) 1519–1524. (2001) 601–606. [168] H. Yim, R. Haselbeck, W. Niu, C. Pujol-Baxley, A. Burgard, J. Boldt, J. Khandurina, [140] W.J.H. van Berkel, N.M. Kamerbeek, M.W. Fraaije, Flavoprotein monooxygenases, J.D. Trawick, R.E. Osterhout, R. Stephen, J. Estadilla, S. Teisan, H.B. Schreyer, a diverse class of oxidative biocatalysts, J. Biotechnol. 124 (2006) 670–689. S. Andrae, T.H. Yang, S.Y. Lee, M.J. Burk, S. Van Dien, Metabolic engineering of [141] A.C. Rosenzweig, C.A. Frederick, S.J. Lippard, P. Nordlund, Crystal structure of a Escherichia coli for direct production of 1,4-butanediol, Nat. Chem. Biol. 7 (2011) bacterial non-haem iron hydroxylase that catalyses the biological oxidation of 445–452. methane, Nature 366 (1993) 537–543. [169] S.J. Berrios-Rivera, A.M. Sanchez, G.N. Bennett, K.-Y. San, Effect of different levels [142] H. Basch, K. Mogi, D.G. Musaev, K. Morokuma, Mechanism of the methane → of NADH availability on metabolite distribution in Escherichia coli fermentation in methanol conversion reaction catalyzed by methane monooxygenase: a density minimal and complex media, Appl. Microbiol. Biotechnol. 65 (2004) 426–432. functional study, J. Am. Chem. Soc. 121 (1999) 7249–7256. [170] J. Moon, Z.L. Liu, Engineered NADH-dependent GRE2 from Saccharomyces cere- [143] T.J. Lawton, A.C. Rosenzweig, Methane-oxidizing enzymes: an upstream problem visiae by directed enzyme evolution enhances HMF reduction using additional in biological gas-to-liquids conversion, J. Am. Chem. Soc. 138 (2016) 9327–9340. cofactor NADPH, Enzym. Microb. Technol. 50 (2012) 115–120. [144] T.J. Smith, J.C. Murrell, Mutagenesis of soluble methane monooxygenase, [171] J.K.B. Cahn, C.A. Werlang, A. Baumschlager, S. Brinkmann-Chen, S.L. Mayo, Methods Enzymol. 495 (2011) 135–147. F.H. Arnold, A general tool for engineering the NAD/NADP cofactor preference of [145] H. Olteanu, R. Banerjee, Human methionine synthase reductase, a soluble P-450 oxidoreductases, ACS Synth. Biol. 6 (2017) 326–333. reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine [172] Y. Wang, K.-Y. San, G.N. Bennett, Cofactor engineering for advancing chemical synthase activation, J. Biol. Chem. 276 (2001) 35558–35563. biotechnology, Curr. Opin. Biotechnol. 24 (2013) 994–999. [146] B. Fox, C.T. Walsh, Mercuric reductase. Purification and characterization of a [173] R. Woodyer, W.A. van der Donk, H. Zhao, Relaxing the nicotinamide cofactor transposon-encoded flavoprotein containing an oxidation-reduction-active dis- specificity of phosphite dehydrogenase by rational design, Biochemistry 42 (2003) ulfide, J. Biol. Chem. 257 (1982) 2498–2503. 11604–11614. [147] P. Lian, H.-B. Guo, D. Riccardi, A. Dong, J.M. Parks, Q. Xu, E.F. Pai, S.M. Miller, [174] M. Katzberg, N. Skorupa-Parachin, M.-F. Gorwa-Grauslund, M. Bertau, D.-Q. Wei, J.C. Smith, H. Guo, X-ray structure of a Hg2+ complex of mercuric Engineering cofactor preference of ketone reducing biocatalysts: a mutagenesis reductase (MerA) and quantum mechanical/molecular mechanical study of Hg2+ study on a γ-diketone reductase from the yeast Saccharomyces cerevisiae serving as transfer between the C-terminal and buried catalytic site cysteine pairs, an example, Int. J. Mol. Sci. 11 (2010) 1735–1758. Biochemistry 53 (2014) 7211–7222. [175] J.T. Wu, L.H. Wu, J.A. Knight, Stability of NADPH: effect of various factors on the [148] K. Okamoto, T. Kusano, T. Nishino, Chemical nature and reaction mechanisms of kinetics of degradation, Clin. Chem. 32 (1986) 314–319. the molybdenum cofactor of xanthine oxidoreductase, Curr. Pharm. Des. 19 [176] G.A. Khoury, H. Fazelinia, J.W. Chin, R.J. Pantazes, P.C. Cirino, C.D. Maranas, (2013) 2606–2614. Computational design of Candida boidinii xylose reductase for altered cofactor [149] K. Fukuyama, Structure and function of plant-type ferredoxins, Photosynth. Res. specificity, Protein Sci. 18 (2009) 2125–2138. 81 (2004) 289–301. [177] S. Brinkmann-Chen, T. Flock, J.K.B. Cahn, C.D. Snow, E.M. Brustad, J.A. McIntosh, [150] G.A. Ziegler, C. Vonrhein, I. Hanukoglu, G.E. Schulz, The structure of Adrenodoxin P. Meinhold, L. Zhang, F.H. Arnold, General approach to reversing ketol-acid re- reductase of mitochondrial P 450 systems: electron transfer for steroid biosynth- ductoisomerase cofactor dependence from NADPH to NADH, Proc. Natl. Acad. Sci. esis, J. Mol. Biol. 289 (1999) 981–990. U. S. A. 110 (2013) 10946–10951. [151] E.J. McKenna, M.J. Coon, Enzymatic ω-oxidation: IV. Purification and properties [178] D. Agren, M. Stehr, C.L. Berthold, S. Kapoor, W. Oehlmann, M. Singh, of the ω-hydroxylase of Pseudomonas oleovorans, J. Biol. Chem. 245 (1970) G. Schneider, Three-dimensional structures of apo- and holo-L-alanine dehy- 3882–3889. drogenase from Mycobacterium tuberculosis reveal conformational changes upon [152] I.F. Sevrioukova, T.L. Poulos, I.Y. Churbanova, Crystal structure of the coenzyme binding, J. Mol. Biol. 377 (2008) 1161–1173. Putidaredoxin reductase·Putidaredoxin electron transfer complex, J. Biol. Chem. [179] A. Lerchner, A. Jarasch, A. Skerra, Engineering of alanine dehydrogenase from 285 (2010) 13616–13620. Bacillus subtilis for novel cofactor specificity, Biotechnol. Appl. Biochem. 63 (2016) [153] J. Sancho, Flavodoxins: sequence, folding, binding, function and beyond, Cell. 616–624. Mol. Life Sci. 63 (2006) 855–864. [180] B. Laadan, J.R.M. Almeida, P. Rådström, B. Hahn-Hägerdal, M. Gorwa-Grauslund, [154] H.A. Relyea, W.A. van der Donk, Mechanism and applications of phosphite de- Identification of an NADH-dependent 5-hydroxymethylfurfural-reducing alcohol hydrogenase, Bioorg. Chem. 33 (2005) 171–189. dehydrogenase in Saccharomyces cerevisiae, Yeast 25 (2008) 191–198. [155] Y. Zou, H. Zhang, J.S. Brunzelle, T.W. Johannes, R. Woodyer, J.E. Hung, N. Nair, [181] P. Rellos, J. Ma, R.K. Scopes, Alteration of substrate specificity of Zymomonas W.A. van der Donk, H. Zhao, S.K. Nair, Crystal structures of phosphite dehy- mobilis alcohol dehydrogenase-2 using in vitro random mutagenesis, Protein Expr. drogenase provide insights into nicotinamide cofactor regeneration, Biochemistry Purif. 9 (1997) 83–90. 51 (2012) 4263–4270. [182] K. Vedha-Peters, M. Gunawardana, J.D. Rozzell, S.J. Novick, Creation of a broad- [156] S.R. Thomas, P.M. McTamney, J.M. Adler, N. Laronde-Leblanc, S.E. Rokita, Crystal range and highly stereoselective D-amino acid dehydrogenase for the one-step structure of iodotyrosine deiodinase, a novel flavoprotein responsible for iodide synthesis of D-amino acids, J. Am. Chem. Soc. 128 (2006) 10923–10929. salvage in thyroid glands, J. Biol. Chem. 284 (2009) 19659–19667. [183] H. Akita, K. Doi, Y. Kawarabayasi, T. Ohshima, Creation of a thermostable + [157] J.R. Warner, S.D. Copley, Pre-steady-state kinetic studies of the reductive deha- NADP -dependent D-amino acid dehydrogenase from Ureibacillus thermosphaericus logenation catalyzed by tetrachlorohydroquinone dehalogenase, Biochemistry 46 strain A1 meso-diaminopimelate dehydrogenase by site-directed mutagenesis, (2007) 13211–13222. Biotechnol. Lett. 34 (2012) 1693–1699. [158] F. Velazquez, S.Y. Peak-Chew, I.S. Fernandez, C.S. Neumann, R.R. Kay, [184] T.W. Johannes, R.D. Woodyer, H. Zhao, Directed evolution of a thermostable Identification of a eukaryotic reductive dechlorinase and characterization of its phosphite dehydrogenase for NAD(P)H regeneration, Appl. Environ. Microbiol. 71 mechanism of action on its natural substrate, Chem. Biol. 18 (2011) 1252–1260. (2005) 5728–5734. [159] V. Romanov, R.P. Hausinger, NADPH-dependent reductive ortho dehalogenation [185] X.G. Wang, K.L. Britton, P.J. Baker, S. Martin, D.W. Rice, P.C. Engel, Alteration of of 2,4-dichlorobenzoic acid in Corynebacterium sepedonicum KZ-4 and the amino acid substrate specificity of clostridial glutamate dehydrogenase by site- Coryneform bacterium strainNTB-1 via 2,4-dichlorobenzoyl coenzyme A, J. directed mutagenesis of an active-site lysine residue, Protein Eng. 8 (1995) Bacteriol. 178 (1996) 2656–2661. 147–152. [160] W. Runguphan, J.D. Keasling, Metabolic engineering of Saccharomyces cerevisiae [186] D.J. Maddock, W.M. Patrick, M.L. Gerth, Substitutions at the cofactor phosphate- for production of fatty acid-derived biofuels and chemicals, Metab. Eng. 21 (2014) binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in 103–113. substrate specificity, Protein Eng. Des. Sel. 28 (2015) 251–258.

347