For citation purposes: Elleuche S, Antranikian G. Bacterial group III alcohol dehydrogenases - function, evolution and biotechnological applications. OA Alcohol 2013 Mar 01;1(1):3.

Review

 
Biomedical

Bacterial group III alcohol dehydrogenases–function, evolution and biotechnological applications

S Elleuche1*, G Antranikian1
 

Authors affiliations

(1) Institute of Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, D-21073 Hamburg, Germany

* Corresponding author skander.elleuche@tuhh.de

Abstract

Introduction

Living organisms have developed strategies to cope with alcoholic substrates. Due to the manifold appearance of alcoholic compounds, enzymes that are capable of decomposing or processing such molecules have emerged several times during evolution. These proteins are the so-called alcohol dehydrogenases, a group of ubiquitous enzymes that catalyse the interconversion between alcohols and aldehydes or ketones. In addition to several other co-factors that are used by alcohol dehydrogenases, three non-homologous protein families exclusively use NAD(P)/NAD(P)H. Two of the three protein families have been predominantly and extensively studied in animals; this short review focuses on bacterial group III alcohol dehydrogenases and sheds some light on their functionality, distribution and potential biotechnological applications.

Conclusion

In contrast to other NAD(P)-dependent ADHs, there is only little information on group III ADHs, however, recent investigations have shown some promise for some of these enzymes. The continuous evaluation of such biocatalysts will aid in the development of environment friendly approaches for the production of fine chemicals, which could provide an alternative to traditional petrochemical routes.

Introduction

Alcohol dehydrogenases (ADHs; EC 1.1.1.1 and EC 1.1.1.2), an omnipresent group of enzymes, catalyse the interconversion between alcoholic compounds and their corresponding aldehydes or ketones, an activity essential for different processes in living organisms. These enzymes are abundant in animals, plants, fungi, algae, bacteria and the Archaea domain. They are present in diverse tissues, such as the liver, kidney, gastric mucosa and mammary glands, as well as in micro-organisms thriving in extreme environments, such as solfataric fields or hot vents[1,2,3]. To catalyse the reduction of aldehydes or oxidation of alcohols, ADHs are in need of a co-factor which is used as an electron acceptor. Based on the utilisation of a specific co-factor, it is possible to group these proteins into three different categories: (1) nicotinamide adenine dinucleotide- (NAD-) or nicotinamide adenine dinucleotide phosphate- (NADP-) dependent ADHs, (2) pyrroloquinolinequinone-, haem-group and F420-dependent enzymes and (3) flavin adenine dinucleotide-dependent isozymes[4].

So far, there are three distantly related types of ADHs, which can be grouped as NAD(P)-dependent ADHs. The zinc-containing, long/medium chain ADHs (group I) are the most studied and can be distinguished from the superfamilies of the metal-free, short chain ADHs (group II) and the metal-containing ADHs (group III). Several organisms such as mosquitoes and flies have been described to contain ADHs of all the three different types. In contrast, the human genome comprises of at least seven genes encoding group I ADHs, and a single open reading frame which codes for an ADH belonging to group III[1,5]. Furthermore, the mammalian enzyme horse liver alcohol dehydrogenase is a prototype of the first group, while a group II isozyme was first studied from the fruit fly Drosophila melanogaster[4,6]. The third group of ADHs was first discovered in micro-organisms, and the first isozyme that has been studied in detail is a microbial ADH from the ethanol producing bacterium Zymomonas mobilis[7]. In this review, we have briefly focused on the function, evolution and possible applications of the highly diverse group III of ADHs.

Discussion

The authors have referenced some of their own studies in this review. The protocols of these studies have been approved by the relevant ethics committees related to the institutions in which these studies were performed.

Function of group III ADHs

ADHs fulfil multiple physiological roles and display a wide variety of substrate specificities by exhibiting great substrate promiscuity[4,8]. However, not every isozyme is capable of oxidising short chain alcoholic compounds, such as ethanol. This can also be illustrated by the transcriptional expression profile of ADH encoding genes. While the expression of group II ADH genes is usually induced by ethanol exposure, this is not true for other NAD(P)-dependent ADHs. They often show low affinity and catalytic efficiency towards ethanol as a substrate, indicating again that these enzymes mainly resume versatile roles in the cell without participating in ethanol metabolism[1]. Nevertheless, there is a reaction mechanism that is common for all NAD(P)-dependent ADHs: the reduction of an aldehyde or ketone results in the production of an alcoholic compound and an oxidised co-factor. The regeneration of NAD(P) is often essential for other coupled physiological reactions, especially in microorganisms. In agreement with this reaction, the oxidation of an alcohol results in the production of an aldehyde or ketone and supplies reduced NAD(P)H (Figure 1).

Reaction mechanism of alcohol dehydrogenases. These enzymes catalyse the NAD(P)-dependent interconversion of aldehydes or ketones and enantiomerically pure alcoholic compounds. The complete structure of NAD[+]/NADH is indicated by a red H-atom, while the additional phosphate-group of NADP[+]/NADPH is indicated in green.

ADHs are capable of processing a wide variety of substrates including linear, branched, primary, secondary and circular alcohols in addition to their corresponding aldehydes and ketones. Within the third group of NAD(P)-dependent ADHs, some members are described and characterised as lactaldehyde dehydrogenases, butanol dehydrogenases, 1,3-propanediol (1,3-PD) dehydrogenases, while other isozymes exclusively catalyse the reduction of aldehydes without oxidising alcohols at all[9,10,11,12]. Therefore, it has been speculated, that some group III ADHs are mainly involved in aldehyde reduction in bacterial species rather than alcohol turnover.

The prototype enzyme YqhD from Escherichia coli, shows a rather high affinity to aldehydes coupled with low alcohol oxidation activity, which is in good agreement with group III ADHs from other micro-organisms[8,12,13]. Due to the substrate looseness of group III ADHs, detailed characterisation of various candidate genes is necessary to fully understand their function-evolution relationships. For this reason, it is often hard to disentangle the physiological functions of these enzymes. Isozymes that have mainly been characterised so far include those from the family of Enterobacteriaceae, such as E. coli and Klebsiella pneumoniae and from the hemiascomycetous yeast Saccharomyces cerevisiae. Members from extremophilic micro-organisms, such as (hyper-) thermophilic archaeal and bacterial species, have also been investigated in detail[2,3,9,14,15].

Distantly related members of group III ADHs were encoded as pairs in enterobacterial species and characterised in detail. The enzyme FucO from E. coli is a lactaldehyde: propanediol oxidoreductase catalysing the NADH-dependent reduction of lactaldehyde to produce 1,2-propanediol (1,2-PD)[10,14]. The closely related protein DhaT from K. pneumoniae has been shown to be capable of converting 3-hydroxypropionaldehyde into 1,3-PD, which is important for NAD[+]-production under physiological conditions. Adh3.1 from the plant pathogenic enterobacterium Dickeya zeae, showed highest activity on short chain primary alcohols, but was unable to act on 1,2-PD or 1,3-PD[9,13]. These members are clustered together and named as lactaldehydes: propanediol oxidoreductase-like enzymes or named on the basis of the most studied prototype as DhaT-like enzymes. They can be opposed to the YqhD-like enzymes within the group III ADHs, which are mostly annotated as butanol dehydrogenases. Both groups show functionally overlapping as well as diverse properties (Table 1). Instead of NADH, YqhD-like enzymes exclusively use NADPH as a co-factor and prefer to catalyse the reduction of aldehydes to the oxidation of alcohols. This enzyme is of industrial importance due to its ability to produce 1,3-PD (see below). YqhD from E. coli and Adh3.2 from D. zeae are useful in detoxifying glyoxals[13,16]. It is hypothesised that E. coli YqhD might be involved in protecting the cells from the toxic effects of lipid-oxidation derived aldehydes. Additionally, the physiological role of DhaT from K. pneumoniae is speculated to be the regeneration of NAD[+], which is needed as a co-factor in the oxidative glycerol degradation pathway[12,17].

Table 1

Substrate and cofactor specificities of group III ADHs.

Evolution of group III ADHs

Enzyme evolution is mainly determined by functional convergence and functional diversification[18]. The fact that different non-homologous ADHs are capable of catalysing the interconversion of aldehydes and alcohols, although they are structurally distinct, makes these groups of enzymes an excellent candidate for functional convergent evolution. However, the ability to act on different substrates and/or to be involved in different metabolic pathways of orthologous ADHs within a specific group, may be the result of gene duplications and/or functional diversification.

Group III ADHs are not related to other NAD(P)-dependent ADHs, which means that they arose independently during the course of evolution. The complex and long evolutionary history of this group of enzymes reflects its physiological versatility. While only a single mitochondrial group III ADH has been identified in animal genomes, there have been several enzymes found in prokaryotes, executing multiple physiological roles and exhibiting diverse substrate specificities[1,13]. These enzyme variants might be the result of ancient gene duplications and functional diversification events. Gene duplications followed by functional innovation have been shown to appear very often in enzymes[13,19,20,21]. As a result of functional diversification within the third group of ADHs, these enzymes can be further subdivided and show substantially distinct substrate specificities and functions[10,11,13,14,15,22]. Moreover, it has been hypothesised that group III ADHs were distributed throughout the horizontal gene transfer between the Archaea and Bacteria domains, which might have involved functional diversification and non-orthologous gene displacement[3].

The third group of ADHs contains enzymes that exhibit a size of around 385 amino acid residues (Figure 2). Pairs of the functionally characterised DhaT-like and YqhD-like members of the group III ADHs, show a low identity of approximately 20% over the complete length of the proteins, but show an overall structural similarity. Pairs of these enzymes were found and characterised in several bacterial species, such as K. pneumoniae, E. coli or D. zeae[9,10,13,14,15,23]. However, this was not the case in all of the members of the Enterobacteriaceae family. It was shown that another species of the genus Klebsiella, namely K. oxytoca, does not encode a DhaT-like ADH, but a protein that is >80% identical to the YqhD enzyme from E. coli. Interestingly, this enzyme, YqhD-1, seems to compensate for the loss or the non-existence of the DhaT-like ADH by exhibiting low activity towards several alcoholic compounds[24]. Although metal-ion containing ADHs appear alike in their tertiary structure, they differ in their quaternary structure. Dimeric, tetrameric, octameric and decameric states have been described in this superfamily[9,10,11,22,25,26]. Moreover, there is a difference in the presence of the metal-ion in the conserved catalytic region of the group III ADHs. While iron or zinc were predominantly identified as catalytic ions, the recently solved structure of Adh3 from the gram-positive bacterium Oenococcus oeni, revealed the presence of a nickel ion in the dimeric structure of this protein[25]. Another striking difference was found within the primary structure of the order Lactobacillales (including O. oeni), where the coordinating amino acid residues are asparagine, two histidines and an atypical glutamine which is replaced by a third histidine in well-characterised structures from other members of the group III ADHs (Figure 2). In addition, the metal ions of DhaT-like members of the group III ADHs were bound more tightly in comparison to the YqhD-like isozymes[9,24,25]. Nevertheless, the great diversity within this group of enzymes complicates the functional annotation of newly identified sequences that are neither expressed nor characterised in detail.

Schematic illustration depicting the gene structure of group III ADHs including a multiple sequence alignment of the co-factor binding and metal-ion coordinating region. ClustalX was used with the following sequences: D. zeae Adh3.1 (HF546061), Adh3.2 (HF546062); E. coli FucO (AAA23825.1), YqhD (NP_417484.1); K. pneumonia DhaT (YP_005956552.1), KpYqhD (ABR78827.1); O. oeni Adh3 (HE974350) and Z. mobilis ZmAdh2 (BAF76066.1). A black line indicates the co-factor binding region and asterisks highlight the conserved amino acid residues involved in metal-ion coordination. Protein lengths are indicated beside the alignment. Members of the DhaT-like proteins are boxed in light blue and a light grey box illustrates members of the YqhD-like isozymes.

Applications of group III ADHs

The history of the biotechnological applications of ADHs commenced more than 100 years ago when noble prize laureate Eduard Buchner established the process of alcohol fermentation without the utilisation of yeast cells[27]. Group III ADHs do not play a role in the production of alcoholic beverages, but are mainly of industrial importance as biocatalysts for the synthesis of fine chemicals.

A well-known example for the industrial utilisation of group III ADHs in the production of chemical compounds (e.g. 1,3-PD), is the application of DhaT and YqhD in recombinant E. coli. This small molecule can be used as a monomer for the production of a variety of products, such as synthetic polymers, drugs, fibres or cosmetics[28]. A lot of effort has been made to generate multi-mutated, engineered, expression strains to increase the titre of 1,3-PD produced from glycerol. Moreover, it has been shown that ADHs YqhD and FucO are important for the production of 1,2-PD from glycerol in E. coli with YqhD exhibiting the additional advantage of reducing methylglyoxal to acetol; the latter compound has the ability of being converted to 1,2-PD by another enzyme, namely the D-aminopropanol dehydrogenase GldA[8].

As mentioned above, the physiological role of YqhD might be to act as an aldehyde scavenger, which prompted Lee and Park[29] to develop another interesting application of YqhD. In a recent publication, these authors came up with a suicidal vector-cloning system which is based on the toxic effects of butanal and propanal in the host E. coli[29]. It has been shown that E. coli is sensitive to butanal and other aldehydes, when yqhD-encoding genes are overexpressed in a recombinant form and these aldehydes are reduced to give alcoholic compounds[13,16,29]. In the presented system, the lacZ-gene in vector pUC19 is replaced by a yqhD-variant with a preceding multiple cloning site (MCS). The expression of yqhD leads to lethality when E. coli transformants are incubated on butanal- or propanal-containing plates. The insertion of a foreign DNA-fragment into the MCS interferes with the expression of yqhD, which results in transformants that survive on aldehyde-containing growth plates[29].

Conclusion

ADHs are a family of proteins with a complex and interesting evolutionary history. This has led to the generation of enzymes with broad substrate promiscuity. These proteins can be involved in either a variety of distinct pathways or an exclusive catalysation of well-balanced specific reactions, often producing enantiomerically pure products. In contrast to other NAD(P)-dependent ADHs, there is only little information on group III ADHs so far. However, some recent investigations have proven the promising applicability of some of these enzymes. The continuous evaluation of such biocatalysts will open new avenues for environmentally friendly production of fine chemicals, which can effectively compete with traditional petrochemical routes. Moreover, there is a need for a diverse portfolio of novel, efficient and characterised ADHs to cover the industrial demand of these highly versatile enzymes.

Abbreviations list

ADH, alcohol dehydrogenase; MCS, multiple cloning site; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; 1,2-PD, 1,2-propanediol; 1,3-PD, 1,3-propanediol.

Acknowledgement

The work in our laboratory was funded by the Excellence Cluster in the Excellence Initiative by the State of Hamburg ‘Fundamentals of Synthetic Biological Systems (SynBio)’.

Authors Contribution

All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.

Competing interests

None declared.

Conflict of Interests

None declared.

A.M.E

All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.

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Licensee to OAPL (UK) 2013. Creative Commons Attribution License (CC-BY)

Substrate and cofactor specificities of group III ADHs.

DhaT-like YqhD-like
E. coli FucO14 K. pneumoniae DhaT15 D. zeae Adh3.113 E. coli YqhD8,12 K. pneumoniae YqhD23 D. zeae Adh3.213
Co-factor NAD(H) NAD(H) NAD(H) NADPH NADPH NADPH
Alcohols
Ethanol + - + - n.d. -
Propanol + + + - n.d. -
Butanol n.d. - - - n.d. -
Methanol - - - - n.d. -
Glycerol + - - - n.d. -
1,2-PD + - - - n.d. -
1,3-PD n.d. + - - n.d. -
Aldehydes
Acetaldehyde + n.d. + + n.d. -
Propanal + + + + n.d. -
Butanal n.d. n.d. - + n.d. +
3-HPA n.d. + n.d. + + n.d.
Glyoxal n.d. n.d. - + n.d. +
Methylglyoxal n.d. n.d. + + n.d. +
Glycoaldehyde + n.d. + + n.d. +

NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; n.d., not determined; 1,2-PD, 1,2-propanediol; 1,3-PD, 1,3-propanediol; 3-HPA, 3-hydroxypropionaldehyde.

Keywords