Aldehyde dehydrogenases (ALDHs)

Aldehyde dehydrogenases (ALDHs) are a large family o f enzymes found in all living systems. They convert a broad class o f aldehydes to their corresponding carboxylic acids in what can be considered as an irreversible reaction.

ALDH

R C H O ► RCOOH

NAD (P)" NAD (?) H

ALDH uses NAD^ (Nicotinamide adenine dinucleotide) or NADP^ (Nicotinamide adenine dinucleotide phosphate) as a cofactor (70). In vivo aldehydes are produced as intermediates in the metabolism o f other compounds and ALDHs have evolved that metabolize aldehydes to assist in their removal (71). In mammals ALDHs metabolize a variety o f aldehydes and are ubiquitiously expressed within the body (70, 72). These enzymes all have the same general function and are specific for their roles. Human liver ALDH is one o f the two key enzymes that is responsible for alcohol metabolism (73); firstly alcohol dehydrogenase (ADH) converts ethanol to acetaldehyde and secondly ALDH converts acetaldehyde to acetic acid, which can then be eliminated or used in other metabolic pathways. ALDHs have also been implicated in developmental processes (74) and pheromone metabolism (75) whilst a change in ALDH activity has also been linked to a number o f tumors, including liver, colon and mammary cancers (70). A mutation in the human fatty ALDH has been linked to the Sjogren-Larson Syndrome, an inborn neurologic impairment (76).

Chapter 1 : Introduction

1.9 Structural characteristics o f mammalian ALDHs

ALDHs differ in a range o f physical characteristics including charge, relative substrate preference and inhibitor susceptibility (77-78). Mammalian ALDHs have been purified and characterised from a variety o f tissues including liver, brain, stomach, cornea, lens and testis; cDNAs o f ALDHs from some o f these sources have been used in bacterial expression systems (70). The enzyme functions as either homodimers or

homotetramers. The major constitutive cytosolic and mitochondrial ALDHs o f liver are tetramers composed o f identical sub-units o f approximately 55 kDa which contain about 500 amino acid residues. The major constitutive ALDHs o f cornea and stomach function as homodimers comprising ~50 kDa monomers containing about 450 amino acid residues (77).

1.10 Classification of mammalian ALDHs

In 1988 the primary sequences o f five mammalian ALDHs were known: the human, and horse cytosolic and mitochondrial pairs and the rat tumor/dioxin-inducible ALDH. Even with the knowledge o f the known sequences it was apparent that 3 different classes could be identified and the enzymes have been grouped based on cellular location and substrate specificity (78-84). The 3 major classes o f ALDHs are shown in

Table 1.2.

Table 1.2 Classification of ALDHs

Class Cell localization Substrate specificity Ref

1 Cytosol RAL 85-93

2 Mitochondria acetaldehyde 100-110

3 microsomes and

cytosol

aromatic, fatty aldehydes and tumor associated aldehydes

119-126

1.10.1 Class 1

Cytosolic ALDHs show a high specificity for RALs (85-93). Studies on mouse embryos show that RAL-specific ALDH activity co-localises with high concentrations o f RA in the developing spinal chord (74) and in the mouse embryonic retina (94). RAL-specific ALDHs can be divided into two groups on the basis o f their amino acid sequence and pi values (93). The archetypal class 1 ALDH is found predominantly in the liver o f higher vertebrates including horse, bovine, sheep and man and has a pi o f -

5.2. Two retinal-specific variants o f ALDHl (retinaldehyde dehydrogenase types 1 and 2 (Raldh-1 and 2)) have been characterised in the mouse and rat. Raldh-1 is about 90% identical to the class 1 enzymes, with a high pi o f ~8.3 (88, 80, 82, 94) and effectively converts both 9-cis (9C) and all-trans RAL to the corresponding RA (95-96). The other enzyme Raldh-2 is only about 70% identical to the classical ALDHl enzymes, has a pi of -5 .1 , and has a specific role o f oxidizing RAL (81, 97-98).

1.10.2 Class 2

ALDH such as the isoform found in liver, ALDH2, is produced with a leader sequence that is targeted to the mitochondrial matrix, has a pi o f -5 .0 with a molecular mass around 55 kDa. ALDHl and ALDH2 are both tetrameric, with individual sub-units comprising 499-500 amino acid residues that show 69% sequence identity. The presence o f an alcohol-sensitive phenotype mapping to the human ALDH2 locus (99- 104) strongly indicates that the major biological function o f ALDH2 is to metabolize ethanol-derived acetaldehyde. In addition, human ALDH2 does not metabolise all-trans

RAL (100). The alcohol sensitivity in Orientals is associated with the genetic

deficiency o f ALDH2 caused by a G-A point mutation in exon 12, which changes Glu to Lys at position 487 (105-6). The accumulation o f toxic acetaldehyde occurs after people with this phenotype drink ethanol. This leads to alcohol-associated symptoms such as facial flushing and nausea (107-108). Unexpectedly people possessing the inactive lysine-variant were found to possess the DNA coding for both the active glutamate enzyme and the inactive lysine enzyme (109-111).

Disulfiram used in aversion therapy for alcoholism leads to inhibition o f hepatic ALDH activity (112). Ingestion o f ethanol by an individual on disulfiram therapy causes acetaldehyde to accumulate, which leads to several undesirable effects including nausea, vomiting and facial flushing (112-113). Metabolites o f disulfiram, have been shown to be potent inhibitors o f recombinantly expressed human and mitochondrial and cytosolic ALDH (114-116) and ALDH activity in rat liver mitochondria (117).

C h apter 1 In trod u ction

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Fig. 1.4 Structure o f rat ALDH 3

A ribbon diagram o f a ALDH 3 (rat) monomer using the Rasmol program with labels for domains and backbone (green), the active site thiol group Cys 243 (yellow) that is part of the catalytic domain and the relative location o f the NAD (P)^ cofactor (indigo).

FlG.1.5 Structure o f bovine ALDH 2

A ribbon diagram o f A LDH 2 (bovine) monom er using the Rasmol program with labels for domains (green), the relative location o f the NAD^ cofactor (indigo), and the following conserved am ino acid residues C301 (top yellow), C302 (bottom yellow), E268 (top pink), E399 (bottom pink), N 169 (purple) and K 192 (blue) in the active site region.

C hapter 1: Introduction

enzyme is not normally expressed in rodent liver, but is found in other organs including stomach and cornea (70). The enzyme is described as a tumor-associated ALDH, since exposure to carcinogens activates the ALDHS gene in the liver (70). The ALDHS isozyme is induced by polycyclic aromatic hydrocarbons, not all o f which are classified as carcinogens (120-126).