Non-P450 aldehyde oxidizing enzymes: the aldehyde dehydrogenase superfamily Satori A Marchitti, Chad Brocker*, Dimitrios Stagos*, and Vasilis Vasiliou��� University of Colorado Health Sciences Center, Molecular Toxicology & Environmental Health Sciences Program, Department of Pharmaceutical Sciences, Denver, Colorado 80262, USA Abstract Background���Aldehydes are highly reactive molecules. While several non-P450 enzyme systems participate in their metabolism, one of the most important is the aldehyde dehydrogenase (ALDH) superfamily, composed of NAD(P)+-dependent enzymes that catalyze aldehyde oxidation. Objective���This article presents a review of what is currently known about each member of the human ALDH superfamily including the pathophysiological significance of these enzymes. Methods���Relevant literature involving all members of the human ALDH family was extensively reviewed, with the primary focus on recent and novel findings. Conclusion���To date, 19 ALDH genes have been identified in the human genome and mutations in these genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases, including Sj��gren-Larsson syndrome, type II hyperprolinemia, ��-hydroxybutyric aciduria and pyridoxine-dependent seizures. ALDH enzymes also play important roles in embryogenesis and development, neurotransmission, oxidative stress and cancer. Finally, ALDH enzymes display multiple catalytic and non-catalytic functions including ester hydrolysis, antioxidant properties, xenobiotic bioactivation and UV light absorption. Keywords aldehyde dehydrogenase aldehyde metabolism ALDH 1. Introduction Aldehydes are generated from a wide variety of endogenous and exogenous precursors during numerous physiological processes, including the biotransformation of endogenous compounds such as amino acids, neurotransmitters, carbohydrates, and lipids [1���3]. More than 200 aldehyde species arise from the oxidative degradation of cellular membrane lipids, also known as lipid peroxidation (LPO), including 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) [4]. Amino acid catabolism generates several aldehyde intermediates, including glutamate ��-semialdehyde, while neurotransmitters, such as gamma-aminobutyric acid (GABA), serotonin, noradrenaline, adrenaline, and dopamine, also give rise to aldehyde metabolites [2,5]. Xenobiotics and drugs ��� including ethanol, which generates acetaldehyde, ���Author for correspondence: University of Colorado Health Sciences Center, Molecular Toxicology & Environmental Health Sciences Program, Department of Pharmaceutical Sciences, 4200 East Ninth Avenue, C238, Denver, Colorado 80262, USA, Tel: +1 303 315 6153 Fax: +1 303 315 0274 E-mail: Vasilis.Vasiliou@uchsc.edu. *Both authors contributed equally to this manuscript Declaration of interest The authors state no conflict of interest and have received no payment in preparation of this manuscript. NIH Public Access Author Manuscript Expert Opin Drug Metab Toxicol. Author manuscript available in PMC 2009 March 19. Published in final edited form as: Expert Opin Drug Metab Toxicol. 2008 June 4(6): 697���720. doi:10.1517/17425250802102627. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

and the anticancer drugs cyclophosphamide (CP) and ifosfamide, which generate acrolein ��� are important aldehyde precursors. Various aldehydes, including formaldehyde, acetaldehyde and acrolein, are also ubiquitous in the environment and are present in smog, cigarette smoke and motor vehicle exhaust. Aldehydes are also used or generated in a wide variety of industrial applications including in the production of resins, polyurethane and polyester plastics. In addition, numerous dietary aldehydes, including citral and benzaldehyde, naturally exist or are approved additives in various foods where they impart flavor and odor. While some aldehydes play vital roles in normal physiological processes, including vision, embryonic development, and neurotransmission, many are cytotoxic and carcinogenic [6]. Aldehydes are strong electrophilic compounds with terminal carbonyl groups, making them highly reactive, and ��,��-unsaturated aldehydes, such as 4-HNE and acrolein, also contain a second electrophile at the ��-carbon. Unlike free radicals, aldehydes are relatively long-lived and not only react with cellular components in the vicinity of their formation but, through diffusion or transportation, also affect targets some distance away [4]. Aldehydes form adducts, believed to be the primary mechanism underlying their toxicity, with various cellular targets including glutathione (GSH), nucleic acids, and protein amino acids leading to impaired cellular homeostasis, enzyme inactivation, DNA damage, and cell death [7,8]. Aldehydes are detoxified primarily through reductive and oxidative Phase I enzyme-catalyzed reactions, including the non-P450 aldehyde reduction enzyme systems alcohol dehydrogenase (ADH), aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR), and aldehyde oxidation enzyme systems xanthine oxidase (XO), aldehyde oxidase (AOX) and aldehyde dehydrogenase (ALDH) (Figure 1). The ALDH superfamily catalyzes the oxidation of numerous aldehyde substrates and, while other enzymes metabolize aldehydes, these enzymes play a particularly critical role in the cellular protection against these toxic species, as evidenced by the fact that mutations and polymorphisms in ALDH genes (leading to perturbations in aldehyde metabolism) are the molecular basis of several disease states and metabolic anomalies [2]. The present paper comprehensively reviews the 19 human ALDH proteins. 2. The ALDH superfamily The human ALDH superfamily consists of 19 putatively functional genes with distinct chromosomal locations (Figure 2)[9]. A standardized gene nomenclature system based on divergent evolution and amino acid identity was established for the ALDH superfamily in 1998 [10]. The ALDH enzymes catalyze the NAD(P)+-dependent irreversible oxidation of a wide spectrum of endogenous and exogenous aldehydes (Table 1). ALDH proteins are found in all subcellular regions including cytosol, mitochondria, endoplasmic reticulum and nucleus, with several found in more than one compartment. ALDH isozymes found in organelles other than cytosol possess leader or signal sequences that allow their translocation to specific subcellular regions [11]. After translocation or import, mitochondrial sequences may be removed (resulting in shorter mature proteins), while microsomal and nuclear signals remain intact [12,13]. Most of the ALDHs have a wide tissue distribution and display distinct substrate specificity [2,14]. Generally regarded as detoxification enzymes, ALDHs serve to protect cells from the effects of aldehydes by oxidizing them to their respective carboxylic acids (Figure 1). This is evident from several studies in which an ALDH has been shown to protect against aldehyde-induced cytotoxicity [13]. However, the most compelling evidence relies on the observation that mutations and polymorphisms in ALDH genes (leading to loss of function) are associated with distinct phenotypes in humans and rodents [2,15], including Sj��gren-Larsson syndrome (SLS) [16], type II hyperprolinemia [17], ��-hydroxybutyric aciduria [18], pyridoxine-dependent seizures [19], hyperammonemia [20], alcohol-related diseases [21], cancer [6] and late-onset alzheimer���s disease (AD) [22] (Figure 2). In addition to clinical phenotypes associated with Marchitti et al. Page 2 Expert Opin Drug Metab Toxicol. Author manuscript available in PMC 2009 March 19. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

mutations in ALDH genes, transgenic knockout mice have suggested a pivotal role of ALDHs in physiological functions and processes, such as embryogenesis and development [23,24]. Aside from their role in aldehyde detoxification, many ALDH enzymes possess multiple additional catalytic and non-catalytic functions (Table 1). Indeed, several ALDHs are known to catalyze ester hydrolysis [25] and act as binding proteins for various endogenous (e.g., androgen, cholesterol and thyroid hormone) and exogenous (e.g., acetaminophen) compounds [2]. Additionally, ALDH enzymes may have important antioxidant roles including the production of NAD(P)H [26,27], the absorption of UV light [28,29] and the scavenging of hydroxyl radicals via cysteine and methionine sulfhydryl groups [30]. ALDH enzymes share a number of highly conserved residues necessary for catalysis and cofactor binding [31���36]. The invariant catalytic cysteine Cys-302 (numbering based on the mature human ALDH2 protein), Glu-268, Gly-299, and Asn-169 are all essential for catalysis. Gly-245 and Gly-250 are essential residues of the ALDH Rossmann fold (GxxxxG) necessary for cofactor binding. In addition, Lys-192, Glu-399, and Phe-401 are believed to be integral for cofactor binding and may facilitate catalysis. Crystal structures of mammalian ALDH enzymes have revealed that each subunit contains three domains, namely an NAD(P)+ cofactor- binding domain, a catalytic domain, and a bridging domain [31,32]. At the interface of these domains lies a funnel passage leading to the catalytic pocket. The upper portion of the funnel, composed of residues from all three domains, is believed to confer the required ALDH specificity toward particular aldehyde substrates. The lower portion of the funnel, made up of highly conserved residues from both the cofactor and catalytic domains, appears to be the catalytic site where hydride transfer from substrate to cofactor takes place. Based on crystallographic structures of ALDH enzymes, a catalytic mechanism has been proposed involving acylation, followed by deacylation (Figure 3)[31,32,37���39]. Briefly, cofactor binding results in a conformational change and activation of the catalytic Cys-302 nucleophile, which is positioned by Gly-299 [32]. Cys-302 then attacks the aldehydic function of the substrate and forms an oxyanion thiohemiacetal intermediate, stabilized in part by Asn-169 [31]. The negatively-charged oxygen of the oxyanion intermediate then facilitates hydride transfer to the cofactor, resulting in the formation of a thioacylenzyme intermediate. Hydrolysis of the thioaceylenzyme and release of carboxylic acid product takes place via Glu-268, which acts as a general base by activating the hydrolytic water after hydride transfer. For most ALDHs, the reduced cofactor is believed to dissociate from the enzyme last. However, one human ALDH, namely ALDH6A1, is CoA-dependent and has a slightly different catalysis mechanism in which the reduced cofactor is released prior to the deacylation step and yields a CoA ester product instead of a free acid [40]. 3. ALDH1A1 ALDH1A1 encodes a homotetramer ubiquitously distributed in the adult epithelium of various organs including testis, brain, eye lens, liver, kidney, lung and retina [41,42]. ALDH1A1 is one of three highly conserved cytosolic isozymes (see ALDH1A2 and ALDH1A3) that catalyze the oxidation of the retinol metabolite, retinal (retinaldehyde), to retinoic acid (RA) [43,44]. ALDH1A1 has high affinity for the oxidation of both all-trans-(Km 0.1 ��M) and 9-cis-retinal [45]. RA regulates gene expression by serving as a ligand for nuclear RA receptors (RAR) and retinoid X receptors (RXR). Its synthesis is critical for normal growth, differentiation, development and maintenance of adult epithelia in vertebrate animals [46]. In retinoid- dependent tissues (including the retina), retinal-oxidizing ALDHs have been shown to exhibit differential expression patterns during rodent organogenesis [47���49], indicating that RA signaling is necessary for embryogenesis [50,51]. The in vivo role of ALDH1A1 in RA Marchitti et al. Page 3 Expert Opin Drug Metab Toxicol. Author manuscript available in PMC 2009 March 19. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

synthesis is evidenced by the fact that, while Aldh1a1���/��� mice are viable and have normal morphology of the retina, the livers of Aldh1a1���/��� mice display reduced RA synthesis and increased serum retinal levels after retinol treatment [52,53]. Interestingly, Aldh1a1���/��� mice are protected against both diet-induced obesity and insulin resistance, suggesting that retinal may transcriptionally regulate the metabolic response to high-fat diets and that ALDH1A1 may be a candidate gene for therapeutic targeting [54]. In cultured hepatocytes, supression of ALDH1A1 reduces both the omega oxidation of free fatty acids and the production of reactive oxygen species (ROS) [55]. RXR�����/��� mice display decreased liver ALDH1A1 levels, suggesting that RA binding is an activating factor in ALDH1A1 gene expression [56]. The androgen receptor may also be involved in regulating levels of ALDH1A1 [57], which is known to be an androgen binding protein [58]. RA is required for testicular development and ALDH1A1 is absent in genital tissues of humans with androgen receptor-negative testicular feminization [57,59]. In the human brain, ALDH1A1 is highly expressed in dopaminergic neurons [60], which are known to require RA for their differentiation and development [61]. In these neurons, ALDH1A1 is under the control of Pitx3, a homeodomain transcription factor [61] that may regulate the specification and maintenance of distinct populations of dopaminergic neurons through ALDH1A1 upregulation [62]. Decreased levels of ALDH1A1 occur in dopaminergic neurons of the substantia nigra in Parkinson���s disease (PD) patients [63] and in those of the ventral tegmental area in schizophrenic patients [60]. In the central nervous system (CNS), monoamine oxidase (MAO) metabolizes dopamine to its aldehyde metabolite, 3,4- dihydroxyphenylacetaldehyde (DOPAL). Increasing evidence suggests that DOPAL may be neurotoxic, and its accumulation may lead to cell death associated with neurological pathologies [5]. ALDH1A1 may play a critical role in maintaining low intraneuronal levels of DOPAL by catalyzing its metabolism to 3,4-dihydroxyphenylacetic acid (DOPAC) [5,60]. ALDH1A1 is one of 139 genes that are differentially expressed in primary human hematopoietic stem cells (HSCs) and, through the production of RA, ALDH1A1 has been shown to promote their differentiation [64,65]. These data suggest that ALDH1A1 inhibition could potentially be used for the therapeutic amplification of HSCs. ALDH1A1 is one of the major enzymes involved in the metabolism of the ethanol metabolite, acetaldehyde (Km 50 ��� 180 ��M), to which many of the deleterious effects of ethanol are attributed [66]. Indeed, low ALDH1A1 activity may account for alcohol sensitivity in some Caucasian populations [67,68]. Decreased levels of ALDH1A1 are reported in RXR�����/��� mice, which are more susceptible to alcoholic liver injury [56], while increased ALDH1A1 expression occurs in brains of DBA/2 mice, a mouse strain exhibiting alcohol avoidance [69]. These data in rodents have led to the suggestion that acetaldehyde accumulation in peripheral organs is aversive, while acetaldehyde produced in the brain may be reinforcing. ALDH1A1 also plays a key role in the cellular defense against oxidative stress. Human ALDH1A1 efficiently oxidizes LPO-derived aldehydes, including 4-HNE (Km 17.9 ��M), hexanal (Km 13.4 ��M), and MDA (Km 114.4 ��M) (Figure 4)[41,70]. Using various Aldh1a1���/���mouse models, ALDH1A1 has been demonstrated to play a key role in protecting the mouse eye lens and cornea by detoxifying LPO-derived aldehydes and preventing cataract formation induced by oxidative stresses, including ageing and UV radiation [29]. Similar to other ALDHs, ALDH1A1 may also play an important role in cancer therapeutics (Table 2). ALDH1A1 activity has been reported to decrease the effectiveness of some oxazaphosphorine anticancer drugs, such as CP and ifosfamide, by detoxifying their major active aldehyde metabolites [71]. Indeed, inhibition of ALDH1A1 activity leads to increased toxicity of the major metabolite of CP, 4-hydroperoxycyclophosphamide [72]. Accordingly, Marchitti et al. Page 4 Expert Opin Drug Metab Toxicol. Author manuscript available in PMC 2009 March 19. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

patients with low breast tumor ALDH1A1 levels have been reported to respond to CP-based treatment significantly more often than those with high levels, indicating that ALDH1A1 may be a predictor of the drug���s therapeutic effectiveness [73]. Various noncancerous cells, such as hematopoietic progenitor cells, express relatively high ALDH1A1 levels and thus are relatively resistant to oxazaphosphorine-induced toxicity [74]. ALDH1A1 has also been shown to bind to certain anticancer drugs, including daunorubicin [75] and flavopiridol [76], and is downregulated in certain carcinomas [77]. Recently, ALDH1A1 was found to be downregulated in cell cultures and whole-skin tissue samples from patients with atopic dermatitis, suggesting its use as a potential dermal biomarker of this disease [78]. Aside from aldehyde metabolism, ALDH1A1 possesses esterase activity [79] and has been proposed to be the major enzyme catalyzing the oxidation of 3-deoxyglucosome, a potent glycating agent [80]. In addition, ALDH1A1 binds thyroid hormone [81] and is induced by estrogens [82], suggesting it may be regulated by or involved in hormone signaling. 4. ALDH1A2 ALDH1A2 is a cytosolic homotetramer expressed in various embryonic and adult tissues including intestine, testis, lung, kidney, liver, brain and retina [48,83]. Like ALDH1A1, ALDH1A2 catalyzes the oxidation of both all-trans-retinal and 9-cis-retinal to RA [23]. Compared with other ALDH isozymes [84], ALDH1A2 appears to exhibit the highest specificity (Vmax/Km = 49 nmol��min���1��mg���1����M���1) for all-trans-retinal[43,44]. This property may be due to a unique disordered loop in its active site that binds all-trans-retinal in a distinct manner [85]. ALDH1A2 is involved in several developmental processes and may be a key regulator of RA synthesis in developing tissues [86]. Aldh1��2���/��� mice die in early embryonic stages due to defects in early heart morphogenesis [23,87]. They display a lack of axial rotation, incomplete neural tube closure, reduction of the trunk region [23], and many of the features of human DiGeorge/velocardiofacial syndrome, a disorder characterized by cleft palate, heart abnormalities and learning disabilities [88]. Abnormalities in endothelial cell cycle progression during early vascular development have also been identified in Aldh1��2���/��� embryos [89]. Various animal models have identified Aldh1a2 as a key regulator in the development of numerous tissues including kidney [90], retina [91], lung [92], forebrain [93], pancreas [94], and spinal cord [23]. A significant association between spina bifida in humans and three distinct ALDH1A2 single nucleotide polymorphisms (SNPs), including one silent (A151A c.453A G) and two intronic (rs3784259 and rs3784260), has been found however, their functional significance remains unclear [95]. ALDH1A2 may also play a role in congenital diaphragmatic hernia (CDH), which is associated with chromosomal 15q defects within a region that includes both ALDH1A2 and ALDH1A3[96]. In addition, compounds known to induce CDH have been shown to inhibit ALDH1A2 [97]. ALDH1A2 may play a role in the defense against ethanol toxicity through either acetaldehyde detoxification or the synthesis of RA [98]. While rat ALDH1A2 oxidizes acetaldehyde inefficiently in vitro (Km 0.65 mM) [44], ALDH1A2 induction by lens epithelium-derived growth factor protects cells from ethanol-induced toxicity [99]. In addition, ALDH1A2 is decreased in RXR�����/��� mice, which display increased susceptibility to alcohol-induced liver injury [56]. Marchitti et al. Page 5 Expert Opin Drug Metab Toxicol. Author manuscript available in PMC 2009 March 19. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript