Two forms of NAD-dependent D-mandelate dehydrogenase in Enterococcus faecalis IAM 10071

Two forms of NAD-dependent D-mandelate dehydrogenase (D-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different K(m) and V(max) values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration.     

Recognition site for the side chain of 2-ketoacid substrate in d-lactate dehydrogenase

Replacement of Tyr52 with Val or Ala in Lactobacillus pentosus d-lactate dehydrogenase induced high activity and preference for large aliphatic 2-ketoacids and phenylpyruvate. On the other hand, replacements with Arg, Thr or Asp severely reduced the enzyme activity, and the Tyr52Arg enzyme, the only one that exhibited significant enzyme activity, showed a similar substrate preference to the Tyr52Val and Tyr52Ala enzymes. Replacement of Phe299 with Gly or Ser greatly reduced the enzyme activity with less marked change in the substrate preference. Except for the Phe299Ser enzyme, these mutant enzymes with low catalytic activity consistently stimulated NADH oxidation in the absence of 2-ketoacid substrates. However, the double mutant enzymes, Tyr52Arg/Phe299Gly and Tyr52Thr/Phe299Ser, did not exhibit synergically decreased enzyme activity or the substrate-independent NADH oxidation, but rather increased activities toward certain 2-ketoacid substrates. These results indicate that the coordinative combination of amino acid residues at two positions is pivotal in both the functional recognition of the 2-ketoacid side chain and the protection of the bound NADH molecule from the solvent. Multiplicity in such combinations appears to provide d-LDH-related 2-hydroxyacid dehydrogenases with a great variety of catalytic and physiological functions.     

Conversion of Lactobacillus pentosus D-lactate dehydrogenase to a D-hydroxyisocaproate dehydrogenase through a single amino acid replacement

The single amino acid replacement of Tyr52 with Leu drastically increased the activity of Lactobacillus pentosus NAD-dependent D-lactate dehydrogenase toward larger aliphatic or aromatic 2-ketoacid substrates by 3 or 4 orders of magnitude and decreased the activity toward pyruvate by about 30-fold, converting the enzyme into a highly active D-2-hydroxyisocaproate dehydrogenase.     

Control of the general amino acid permease of Penicillium chrysogenum by transinhibition and turnover

When nitrogen-starved mycelium of Penicillium chrysogenum is incubated with relatively high concentrations of labeled hydrophobic amino acids, influx is followed by efflux of the corresponding labeled α-ketoacid. In spite of the efflux, further transport activity is suppressed. Cell-free extracts contain a transaminase that accepts all those amino acids exhibiting α-ketoacid efflux. Transaminase activity is constitutive but is induced to a 2- to 3-fold higher level during a 2-hr preincubation period with a hydrophobic amino acid. Cycloheximide prevents efflux and also the induction of the transaminase. Cycloheximide itself stimulates a partial decay in transport activity but mycelium preincubated with l-leucine and cycloheximide together retain a greater fraction of the original transport activity than mycelium preincubated with l-leucine alone. The results suggest that transport is regulated partially by transinhibition but a significant part of the substrate-induced decay of transport activity is caused by either (a) the degradation of a permease component (perhaps facilitated by transinhibition), or (b) the induction by the substrate of a regulator protein (perhaps the transaminase).The uptake of labeled substrates by nutrient sufficient mycelium correlates well with lipid solubility of the substrates. This suggests that the nonsaturable uptake observed in these mycelia results from free diffusion of the uncharged species.     

Cytosolic alcohol dehydrogenases from yeast Torulopsis candida]

A comparative study of cell cytosol alcohol dehydrogenase (ADH) from yeast Torulopsis candida IBFM-Y-127 grown on glucose and hexadecane which were the only source of carbon, was made. In both cases ADH had a pH optimum within the range of 7.0--10.0, when various normal primary alcohols (C2--C16) were used. The enzyme was active only in the presence of NAD, which cannot be substituted by NADP. The total activity of ADH decreased approximately 8-fold when the length of hydrocarbon radicals was changed from C2 up to C16. When the cells were grown on hexadecane, only ethyl, n-buthyl, n-amyl and n-hexyl alcohols were active as substrates. The dehydration rate of each alcohol was far lower than that for the cytosol of glucose-grown cells. In the latter case the enzyme activity also decreased with an increase in the alcohol radical from C2 to C6. In all cases studied methyl alcohol and cyclic (cinnamyl alcohol--C8) alcohol were not dehydrated at all. Disc-electrophoresis in polyacrylamide gel, involving gel colouration for the assay of enzyme activity showed that glucose--grown cell cytosol contained three forms of ADH. One of those forms was highly active when short--chain normal primary alcohols were used; this form may be probably regarded as "classical" ADH (EC 1.1.1.1). The two other forms caused intensive dehydration of long-chain alcohols (the best substrates were C7--C10 alcohols for one form and C10--C14 for the others). The two forms of ADH are probably isoenzymes of octanol dehydrogenase (EC 1.1.1.73). Cytosol of cells grown on n-alcane, had a reduced number of ADH forms. The data obtained are discussed in terms of the regulatory role of carbon and energy source (glucose or hexadecane) in the redistribution of alcohol dehydrogenases between structural components of cells (mitochondria) and cytosol.     

Molecular docking simulations of steroid substrates into human cytosolic hydroxysteroid dehydrogenases (AKR1C1 and AKR1C2): insights into positional and stereochemical preferences

AKR1C1 and AKR1C2 are human cytosolic hydroxysteroid dehydrogenases, which play pivotal roles in the metabolism and action of natural and synthetic steroid hormones. The two enzymes are highly homologous, and have distinct positional and stereochemical preferences with various substrates. We performed molecular docking simulations of three steroid substrates, including an androgen (5alpha-dihydrotestosterone, DHT), a progestin (progesterone, PRO), and a synthetic hormone ([7alpha,17alpha]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one or tibolone, TIB), into the active sites of the two enzymes. For each substrate and enzyme pair, the activity inferred by the "productive" docking models (in which the spatial arrangement of the steroid and the cofactor would permit a reaction) matched the experimentally observed positional and stereochemical outcome. These productive conformations were energetically and statistically favored except for TIB and PRO with AKR1C2, where experimentally strong substrate inhibition and low activity were observed, respectively. Results showed that (i) a 3-ketosteroid (DHT) and a 20-ketosteroid (PRO) were reduced by AKR1C1 since the carbonyl groups could occupy the same position by "backwards" binding of steroids; (ii) 3alpha-reduced (DHT) and 3beta-reduced (TIB) products were formed by AKR1C2 since the angular methyl groups of the steroids were inverted by "upside-down" binding of steroids; and (iii) the 3beta- and 3alpha-reduction of DHT by AKR1C1 and AKR1C2, respectively occurred since the steroids employed a "swinging" motion to present opposite faces to the cofactor. Favorable nonproductive modes were observed with all substrates in both enzymes in which the steroid was bound at a "near-entry" position and/or an "in-middle" position, which may influence the reaction coordinate.     

Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis

Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2‐oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20–29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk‐KPR) displayed reductase activity specific for 2‐oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH‐dependent enzymes. Tk‐KPR activity decreased dramatically in the presence of CoA and KPR activity in cell‐free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea.     

Physical training reverses defect in 3-ketoacid CoA-transferase activity in skeletal muscle of diabetic rats

To investigate one potential mechanism whereby physical training improves the plasma concentration of ketone bodies in experimental diabetes mellitus, we measured the activity of 3-ketoacid CoA-transferase, the key enzyme in the peripheral utilization of ketone bodies. Diabetes was induced with streptozotocin (50 mg/kg) and training carried out on a treadmill with a progressive 10-wk program. Diabetes resulted in an increase (P < 0.001) in plasma concentration of beta-hydroxybutyric acid in sedentary rats, which was partly reversed by training (P < 0.001). Diabetes was also associated with a decreased activity of 3-ketoacid CoA-transferase in gastrocnemius muscle. When expressed per total gastrocnemius, training increased the activity of 3-ketoacid CoA-transferase by 66% in nondiabetic rats (P < 0.001) and by 150% in diabetic rats (P < 0.001), the decrease present in diabetic rats being fully reversed by training. Simple linear regression between the log of 3-ketoacid CoA-transferase activity and the log of plasma beta-hydroxybutyric acid levels showed a statistically significant (r = 0.563, P < 0.001) negative correlation. The beneficial effects of training on plasma ketone bodies in diabetic rats are probably explained, at least in part, by an increase in ketone body utilization, mediated by an increase in skeletal muscle 3-ketoacid CoA-transferase activity.     

Characterization of a novel galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T8): the complex formation of beta3Gn-T2 and beta3Gn-T8 enhances enzymatic activity

We characterized a novel member of the beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) gene family, beta3Gn-T8. A recombinant soluble form of beta3Gn-T8 was expressed in Pichia pastoris (P. pastoris), and its substrate specificity was compared with that of beta3Gn-T2. The two enzymes had similar substrate specificities and recognized tetraantennary N-glycans and 2,6-branched triantennary glycans in preference to 2,4-branched triantennary glycans, biantennary glycans, and lacto-N-neotetraose (LNnT), indicating their specificity for 2,6-branched structures such as [Galbeta1-->4GlcNAcbeta1-->2(Galbeta1-->4GlcNAcbeta1-->6)Manalpha1--> 6Man]. Interestingly, when soluble recombinant beta3Gn-T2 and beta3Gn-T8 were mixed, the Vmax/Km value of the mixture was 9.3- and 160-fold higher than those of individual beta3Gn-T2 and -T8, respectively. Sephacryl S-300 gel filtration of the enzymes revealed that apparent molecular weights of each beta3Gn-T2, beta3Gn-T8, and the mixture were 90-160, 45-65, and 110-210 kDa, respectively, suggesting that beta3Gn-T2 and -T8 can form a complex with enhanced enzymatic activity. This is the first report demonstrating that in vitro mixed glycosyltransferases show enhanced enzymatic activity through the formation of a heterocomplex. These results suggested that beta3Gn-T8 and beta3Gn-T2 are cooperatively involved in the elongation of specific branch structures of multiantennary N-glycans.     

Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates

The specificity and reactivity of complement serine proteases D, B, Bb, C2, and C2a were determined using a series of peptide thioester substrates. The rates of thioester hydrolysis were measured using assay mixtures containing the thiol reagent 4,4'-dithiodipyridine at pH 7.5. Each substrate contained a P1 arginine residue, and the effect of various groups and amino acids in the P2, P3, P4, and P5 positions was determined using kcat/Km values to compare reactivities. Among peptide thioesters corresponding to the activation site sequence in B, dipeptide thioesters containing a P2 lysine residue were the best substrates for D. Extending the chain to include a P3 or P4 amino acid resulted in loss of activity, and neither the tripeptide nor the tetrapeptide containing the cleavage sequence of B was hydrolyzed. Overall, D cleaved fewer substrates and was 2-3 orders of magnitude less reactive than C1s against some thioester substrates. C2 and fragment C2a had comparable reactivities and hydrolyzed peptides containing Leu-Ala-Arg and Leu-Gly-Arg, which have the same sequence as the cleavage sites of C3 and C5, respectively. The best substrates for C2 and C2a were Z-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-Leu-Ala-Arg-SBzl, respectively, where Bzl is benzyl. B was the least reactive among these complement enzymes. The best substrate for B was Z-Lys-Arg-SBzl with a kcat/Km value of 1370 M-1 s-1. The catalytic fragment of B, Bb, had higher activity toward these peptide thioester substrates. The best substrate for Bb was Z-Gly-Leu-Ala-Arg-SBzl with a kcat/Km similar to C2a and 10 times higher than the value for B. Both C2a and Bb were considerably more reactive against C3-like than C5-like substrates. Bovine trypsin hydrolyzed thioester substrates with kcat/Km approximately 10(3) higher than the complement enzymes. These thioester substrates for D, B, and C2 should be quite useful in kinetic and active site studies of the purified enzymes.     

Comparison of substrate specificity of alcohol dehydrogenases from human liver, horse liver, and yeast towards saturated and 2-enoic alcohols and aldehydes

The saturated and 2-enoic primary alcohols and aldehydes, ethanol, 1-propanol, 1-butanol, 3-methyl-1-butanol, 1-hexanol, phenylmethanol, 3-phenyl-1-propanol, 2-propen-1-ol, 2-buten-1-ol, 3-methyl-2-buten-1-ol, 2-hexen-1-ol, 3-phenyl-2-propen-1-ol, ethanal, 1-propanal, 1-butanal, 1-hexanal, phenylmethanal, 3-phenyl-1-propanal, 2-propen-1-al, 2-buten-1-al, 2-hexen-1-al, and 3-phenyl-2-propen-1-al, have been compared under uniform conditions as substrates for the alcohol dehydrogenase enzymes from horse and human liver and from yeast. Kinetic constants (K_m arid V) have been measured for each of the substrates with each of the enzymes; equilibrium constants for the various alcohol-aldehyde pairs have also been estimated. The results obtained emphasize the similarities of yeast alcohol dehydrogenase to horse and human liver alcohol dehydrogenase, showing the specificity of yeast alcohol dehydrogenase to be less restricted than formerly believed. In general, the 2-enoic alcohols are better substrates for all three alcohol dehydrogenases than their saturated analogs; on the other hand, saturated aldehydes are better substrates than the 2-enoic aldehydes. Based on these various findings, it is suggested that a more likely candidate than ethanol for the physiological substrate of alcohol dehydrogenase in mammalian systems may well be an unsaturated alcohol, although the wide variety of substrates catalyzed at high rates is not incompatible with a general detoxifying function for alcohols or aldehydes, or both, by alcohol dehydrogenase.     

Kinetic analysis of mouse retinal dehydrogenase type-2 (RALDH2) for retinal substrates

Retinal dehydrogenase (RALDH) isozymes catalyze the terminal oxidation of retinol into retinoic acid (RA) that is essential for embryogenesis and tissue differentiation. To understand the role of mouse type 2 RALDH in synthesizing the ligands (all-trans and 9-cis RA) needed to bind and activate nuclear RA receptors, we determined the detailed kinetic properties of RALDH2 for various retinal substrates. Purified recombinant RALDH2 showed a pH optimum of 9.0 for all-trans retinal oxidation. The activity of the enzyme was lower at 37 degrees C compared to 25 degrees C. The efficiency of conversion of all-trans retinal to RA was 2- and 5-fold higher than 13-cis and 9-cis retinal, respectively. The K(m) for all-trans and 13-cis retinal were similar (0.66 and 0.62 microM, respectively). However, the K(m) of RALDH2 for 9-cis retinal substrate (2.25 microM) was 3-fold higher compared to all-trans and 13-cis retinal substrates. Among several reagents tested for their ability to either inhibit or activate RALDH2, citral and para-hydroxymercuribenzoic acid (p-HMB) inhibited and MgCl(2) activated the reaction. Comparison of the kinetic properties of RALDH2 for retinal substrates and its activity towards various reagents with those of previously reported rat kidney RALDH1 and human liver aldehyde dehydrogenase-1 showed distinct differences. Since RALDH2 has low K(m) and high catalytic efficiency for all-trans retinal, it may likely be involved in the production of all-trans RA in vivo.     

Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.     

Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate bound: substrate recognition, conformational change, and cooperativity

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate, an essential step for the biosynthesis of pantothenate (vitamin B5). Inhibitors of the enzymes of this pathway have been proposed as potential antibiotics or herbicides. Here we present the crystal structure of Escherichia coli KPR in a precatalytic ternary complex with NADP+ and pantoate bound, solved to 2.3 A of resolution. The asymmetric unit contains two protein molecules, each in a ternary complex; however, one is in a more closed conformation than the other. A hinge bending between the N- and C-terminal domains is observed, which triggers the switch of the essential Lys176 to form a key hydrogen bond with the C2 hydroxyl of pantoate. Pantoate forms additional interactions with conserved residues Ser244, Asn98, and Asn180 and with two conservatively varied residues, Asn194 and Asn241. The steady-state kinetics of active site mutants R31A, K72A, N98A, K176A, S244A, and E256A implicate Asn98 as well as Lys176 and Glu256 in the catalytic mechanism. Isothermal titration calorimetry studies with these mutants further demonstrate the importance of Ser244 for substrate binding and of Arg31 and Lys72 for cofactor binding. Further calorimetric studies show that KPR discriminates binding of ketopantoate against pantoate only with NADPH bound. This work provides insights into the roles of active site residues and conformational changes in substrate recognition and catalysis, leading to the proposal of a detailed molecular mechanism for KPR activity.     

Metabolic mayhem caused by 2-ketoacid imbalances

A variety of herbicides act by inhibiting the branched chain amino-acid biosynthetic enzyme, acetolactate synthase (EC 4.1.3.18). The initial consequences of this event are 2-ketoacid accumulation and amino-acid starvation. Recent studies demonstrate that 2-ketoacid imbalances play a significant role in the action of these herbicides; such imbalances may also be important in certain inborn errors of human metabolism.     

Detoxification of promutagenic aldehydes derived from methylpyrenes by human aldehyde dehydrogenases ALDH2 and ALDH3A1

Methylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulpho conjugation to reactive esters, which can induce mutations and tumours. Yet, further oxidation of the alcohol may compete with this toxification. We previously demonstrated that several human alcohol dehydrogenases (ADH1C, 2, 3 and 4) oxidise various benzylic alcohols (derived from alkylated pyrenes) to their aldehydes with high catalytic efficiency. However, all these ADHs also catalysed the reverse reaction, the reduction of the aldehydes to the alcohols, with comparable or higher efficiency. Thus, final detoxification requires elimination of the aldehydes by further biotransformation. We have expressed two human aldehyde dehydrogenases (ALDH2 and 3A1) in bacteria. All pyrene aldehydes studied (1-, 2- and 4-formylpyrene, 1-formyl-6-methylpyrene and 1-formyl-8-methylpyrene) were high-affinity substrates for ALDH2 (K_m = 0.027–0.9 μM) as well as ALDH3A1 (K_m = 0.78–11 μM). Catalytic efficiencies (k_cat/K_m) were higher for ALDH2 than ALDH3A1 by a moderate to a very large margin depending on the substrate. Most important, they were also substantially higher than the catalytic efficiencies of the various ADHs for the reduction the aldehydes to the alcohols. These kinetic properties ensure that ALDHs, and particularly ALDH2, can complete the ADH-mediated detoxification.     

The crystal structure of d-mandelate dehydrogenase reveals its distinct substrate and coenzyme recognition mechanisms from those of 2-ketopantoate reductase

d-Mandelate dehydrogenases (d-ManDHs), belonging to a new d-2-hydroxyacid dehydrogenase family, catalyze the conversion between benzoylformate and d-mandelate using NAD as a coenzyme. We determined the first d-ManDH structure, that of ManDH2 from Enterococcus faecalis IAM10071. The overall structure showed ManDH2 has a similar fold to 2-ketopantoate reductase (KPR), which catalyzes the conversion of 2-ketopantoate to d-pantoate using NADP as a coenzyme. They share conserved catalytic residues, indicating ManDH2 has the same reaction mechanism as KPR. However, ManDH2 exhibits significant structural variations in the coenzyme and substrate binding sites compared to KPR. These structural observations could explain their different coenzyme and substrate specificities.     

Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site

Ketol-acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched-chain amino acids. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration, followed by an NADPH-dependent reduction of a 2-ketoacid. The 2-ketoacid formed by the alkyl migration is not released. Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant. The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2-ketoacids. The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities. Several mutations result in loss of the isomerase activity with retention of reductase activity. However, none of the 17 mutants examined have the isomerase activity only. We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization. Our mechanism explains why the two activities must occur in a single active site without release of a 2-ketoacid and provides a rationale for the requirement for NADPH by the isomerase.     

A new family of 2-hydroxyacid dehydrogenases

The NADH-dependent hydroxypyruvate reductase from cucumber and the pdxB gene product of E. coli display significant homology to E. coli D-3-phosphoglycerate dehydrogenase. In contrast, these proteins do not display much similarity with other oxidoreductases or with other 2-hydroxyacid dehydrogenases in particular. On the basis of their relatedness and the structure of their substrates, these three enzymes constitute a new family of 2-hydroxyacid dehydrogenases distinct from malate and lactate dehydrogenase.     

Aldehyde dehydrogenases and cell proliferation

Aldehyde dehydrogenases (ALDHs) oxidize aldehydes to the corresponding carboxylic acids using either NAD or NADP as a coenzyme. Aldehydes are highly reactive aliphatic or aromatic molecules that play an important role in numerous physiological, pathological, and pharmacological processes. ALDHs have been discovered in practically all organisms and there are multiple isoforms, with multiple subcellular localizations. More than 160 ALDH cDNAs or genes have been isolated and sequenced to date from various sources, including bacteria, yeast, fungi, plants, and animals. The eukaryote ALDH genes can be subdivided into several families; the human genome contains 19 known ALDH genes, as well as many pseudogenes. Noteworthy is the fact that elevated activity of various ALDHs, namely ALDH1A2, ALDH1A3, ALDH1A7, ALDH2*2, ALDH3A1, ALDH4A1, ALDH5A1, ALDH6, and ALDH9A1, has been observed in normal and cancer stem cells. Consequently, ALDHs not only may be considered markers of these cells, but also may well play a functional role in terms of self-protection, differentiation, and/or expansion of stem cell populations. The ALDH3 family includes enzymes able to oxidize medium-chain aliphatic and aromatic aldehydes, such as peroxidic and fatty aldehydes. Moreover, these enzymes also have noncatalytic functions, including antioxidant functions and some structural roles. The gene of the cytosolic form, ALDH3A1, is localized on chromosome 17 in human beings and on the 11th and 10th chromosome in the mouse and rat, respectively. ALDH3A1 belongs to the phase II group of drug-metabolizing enzymes and is highly expressed in the stomach, lung, keratinocytes, and cornea, but poorly, if at all, in normal liver. Cytosolic ALDH3 is induced by polycyclic aromatic hydrocarbons or chlorinated compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, in rat liver cells and increases during carcinogenesis. It has been observed that this increased activity is directly correlated with the degree of deviation in hepatoma and lung cancer cell lines, as is the case in chemically induced hepatoma in rats. High ALDH3A1 expression and activity have been correlated with cell proliferation, resistance against aldehydes derived from lipid peroxidation, and resistance against drug toxicity, such as oxazaphosphorines. Indeed, cells with a high ALDH3A1 content are more resistant to the cytostatic and cytotoxic effects of lipidic aldehydes than are those with a low content. A reduction in cell proliferation can be observed when the enzyme is directly inhibited by the administration of synthetic specific inhibitors, antisense oligonucleotides, or siRNA or indirectly inhibited by the induction of peroxisome proliferator-activated receptor γ (PPARγ) with polyunsaturated fatty acids or PPARγ transfection. Conversely, cell proliferation is stimulated by the activation of ALDH3A1, whether by inhibiting PPARγ with a specific antagonist, antisense oligonucleotides, siRNA, or a medical device (i.e., composite polypropylene prosthesis for hernia repair) used to induce cell proliferation. To date, the mechanisms underlying the effects of ALDHs on cell proliferation are not yet fully clear. A likely hypothesis is that the regulatory effect is mediated by the catabolism of some endogenous substrates deriving from normal cell metabolism, such as 4-hydroxynonenal, which have the capacity to either stimulate or inhibit the expression of genes involved in regulating proliferation.