The Alcohol Dehydrogenases of Saccharomyces Cerevisiae - A Comprehensive Review

MINIREVIEW
The alcohol dehydrogenases of Saccharomyces cerevisiae : a

comprehensive review
Olga de Smidt1,2, James C. du Preez1 & Jacobus Albertyn1
1
Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa; and 2School of Agriculture
and Environmental Sciences, Central University of Technology, Bloemfontein, South Africa
Correspondence: Jacobus Albertyn,

Department of Microbial, Biochemical and
Food Biotechnology, University of the Free
State, PO box 339, Bloemfontein 9300, South
Africa. Tel.: 127 51 401 2223; fax: 127 51
444 3219; e-mail: albertynj.sci@ufs.ac.za
Received 1 December 2007; revised 5 March
2008; accepted 28 March 2008.
First published online 8 May 2008.
DOI:10.1111/j.1567-1364.2008.00387.x
Editor: Patrizia Romano
Keywords
Saccharomyces cerevisiae ; alcohol
dehydrogenase; kinetics; localization;
molecular regulation; function.
Abstract
Alcohol dehydrogenases (ADHs) constitute a large family of enzymes responsible
for the reversible oxidation of alcohols to aldehydes with the concomitant
reduction of NAD1 or NADP1. These enzymes have been identified not only in
yeasts, but also in several other eukaryotes and even prokaryotes. The ADHs of
Saccharomyces cerevisiae have been studied intensively for over half a century. With
the ever-evolving techniques available for scientific analysis and since the completion of the Yeast Genome Project, a vast amount of new information has been
generated during the past 10 years. This review attempts to provide a brief
summary of the wealth of knowledge gained from earlier studies as well as more
recent work. Relevant aspects regarding the primary and secondary structure,
kinetic characteristics, function and molecular regulation of the ADHs in S.
cerevisiae are discussed in detail. A brief outlook also contemplates possible future
research opportunities.
Introduction
Saccharomyces cerevisiae is, without a doubt, the most
important microorganism commercially exploited by humans. No other microorganism has been more intimately
associated with the progress and wellbeing of the human
race than S. cerevisiae and its closely related species. Its
contribution to human progress has been due largely to its
capacity for the ethanolic fermentation of carbohydrate
feedstocks. Two major pathways are involved in the energy
metabolism of S. cerevisiae, namely glycolysis and aerobic
respiration. Ethanol is a key metabolite in energy metabolism, being an end product of glycolysis and ethanolic
fermentation while also serving as a carbon substrate during
aerobic respiration, with the alcohol dehydrogenases
(ADHs) catalysing the interconversion of acetaldehyde and
ethanol.
The ADH systems of various organisms have been
investigated thoroughly in respect to their molecular structure, mode of catalysis and, especially in S. cerevisiae,
physiological significance. During the last century, biochemical analysis of yeast ADH has largely been promoted by the
FEMS Yeast Res 8 (2008) 967978
easy availability of yeast cells. Physiologically, the ADH

reaction in S. cerevisiae and in related species plays a dual
and quite critical role in sugar metabolism. Almost all of the
carbohydrate is used fermentatively, regardless of the availability of oxygen, and a specific ADH isozyme serves to
regenerate the glycolytic NAD1, thereby restoring the redox
balance, through the reduction of acetaldehyde to ethanol.
Under aerobic conditions, respiration of the accumulated
ethanol occurs after depletion of the fermentable sugar,
again by the action of specific ADH isozymes. Thus, in S.
cerevisiae, the ADH reaction links fermentative and respiratory (oxidative) carbon metabolism, allowing the optimal
use of the sugar carbon.
Before the sequencing of the S. cerevisiae genome, completed in April 1996, only five ADHs were known. The
genome sequence, however, revealed the existence of c. 6000
ORFs, including a number of ADHs or sequences possibly
related to these enzymes. Since then, ADH6 and ADH7 have
also been identified and their enzyme products have been
characterized. Considering all the new data available on the
ADHs and the wealth of knowledge gathered over the past
century, a need exists for a comprehensive and extensive
r 2008 University of the Free State

c 2008 Federation of European Microbiological Societies
Journal compilation
Published by Blackwell Publishing Ltd.
968
overview regarding the primary and secondary structure,

mechanisms of function and regulation of these enzymes.
This review constitutes a compilation of the information
gathered since the 1960s pertaining to the molecular biology
and physiological role of the S. cerevisiae ADHs identified to
date.
Classification of the ADHs

ADHs (E.C. 1.1.1.1) are oxidoreductases that catalyse the
reversible oxidation of alcohols to aldehydes or ketones,
with the concomitant reduction of NAD1 or NADP1. ADHs
constitute a large group of enzymes that can be subdivided
into at least three distinct enzyme superfamilies: mediumchain (MDR) and short-chain dehydrogenases/reductases
and iron-activated ADHs (Reid & Fewson, 1994; Kallberg
et al., 2002). The MDR superfamily is divided into different
enzyme families with regard to structural and functional
relationships (Jornvall et al., 2001). The family consists of
enzymes with a subunit size of c. 350 amino acid residues,
dimeric or tetrameric, with two domains in each subunit:
one catalytic and one responsible for binding with the
nucleotide NAD1 or NADP1. Many enzymes of the MDR
family have zinc in their active site and have a sequence
motif known as the zinc-containing ADH signature:
GHEX2GX5(G,A)X2(I,V,A,C,S) (Persson et al., 1993).
The genes encoding classical ADHs include ADH1,
ADH2, ADH3, ADH4 and ADH5 (Lutstorf & Megnet, 1968;
Ciriacy, 1975a; Walton et al., 1986; Feldmann et al., 1994).
Other genes include SFA1, BDH1 (Gonzalez et al., 2000),
BDH2 (YAL061W), SOR1 putative SOR2 (YAL246C),
XDH1, the cinammyl ADH CDH1 and the recently proposed ADH6 (YMR218C) and ADH7 (YCR105W) (Wehner
et al., 1993; van Iersel et al., 1997; Gonzalez et al., 2000;
Larroy et al., 2002a, b).
O. de Smidt et al.
while studying the mechanisms of mitochondrial oxidation

of cytosolic NADH, Overkamp et al. (2000) and Bakker et al.
(2000) obtained evidence that ADH3 was not the sole
mitochondrial ADH. At that time, Adh4p and Ydl114W
were the only probable candidates with no assigned function
or localization. Huh et al. (2003) described the construction
and analysis of a collection of yeast strains expressing fulllength, chromosomally tagged green fluorescent fusion
proteins (GFP). The use of the GFP tag and co-localization
with red fluorescent protein (RFP)-tagged reference proteins allowed them to resolve many related subcellular
compartments with confidence, especially in the case of
proteins for which little functional data existed. They
established that Adh5p was located in the cytoplasm and
that not only Adh3p but Adh4p was also a mitochondrial
enzyme. If this were the case, an explanation is yet to be
provided for the absence of the amino terminal leader
peptide sequence in Adh4p.
Currently, the data as to the localization of Adh6p and
Adh7p are not conclusive, as most information available on
the characterization of these two enzymes was derived from
overexpression studies (Larroy et al., 2002a, b; Valencia et al.,
2003, 2004) or resulted from monitoring expression at the
mRNA level (Petersson et al., 2006).
Primary and secondary structure

Most of the work on yeast ADH genetics is due to the
masterful analysis of Michael Ciriacy (Ciriacy, 1997). Genetic analysis performed on several haploid adh mutants led to
the identification of four unlinked genes, namely ADH1,
ADH2, ADH3 and ADH4. Identification of ADH5 followed
later (Feldmann et al., 1994) and ADH6 and ADH7 with
gene exploration opportunities as a result of the S. cerevisiae
genome project (Gonzalez et al., 2000, Larroy et al.,
2002a, b).
Localization
Eukaryotic cells are organized into a complex network of
membranes and compartments, which are specialized for
various biological functions. A comprehensive knowledge of
the location of proteins within these cellular microenvironments is critical for understanding their functions and
interactions. In the late 1960s, the presence of at least three
NAD-dependent ADHs in bakers yeast was known (Heick
et al., 1969). Two of these enzymes were located in the
cytosol (Adh1p and Adh2p) and fractionation experiments
placed Adh3p in the mitochondrial matrix (van Loon &
Young, 1986).
Drewke & Ciriacy (1988) suggested that it was unlikely
that Adh4p is proteolytically processed as has been demonstrated for Adh3p (Pilgrim & Young, 1987) and that Adh4p
was, therefore, a cytoplasmic enzyme. To date, no other
reports to support this have been published. Furthermore,

Journal compilation
ADH1 (YOL086C) encoding Adh1p

Yeast ADH was the first pyridine nucleotide-dependent
dehydrogenase to be crystallized. Determination of the
primary structure of Adh1p in S. cerevisiae marked the first
case of gene cloning by functional complementation. Physico-chemical methods revealed that the protein had a molecular weight of 150 kDa, that the active enzyme contained
four identical reactive sites and that in all probability it
consisted of four similar, if not identical, polypeptide chains
(Harris, 1964). Yeast Adh1p seems to be a good model for
the study of the stability of complex enzymes, because effects
of the environment on the structure can easily be tested by
measuring the inactivation of reduced and oxidized Adh1p.
Ca21 stabilizes S. cerevisiae Adh1p by preventing the dissociation of the reduced form of the enzyme and by
preventing the unfolding of the oxidized form of the
969
Alcohol dehydrogenases of Saccharomyces cerevisiae
enzyme. It is also possible that Adh1p binds Mg21 in vivo

(de Bolle et al., 1997).
ADH2 (YMR303C) encoding Adh2p

By the early 1980s, the structural genes encoding both
Adh1p (ADH1) and Adh2p (ADH2) were identified genetically (Ciriacy, 1975a), cloned (Williamson et al., 1980, 1981)
and their DNA sequences were determined (Bennetzen &
Hall, 1982; Russell et al., 1983b). High homology was
evident at both the nucleotide sequence level (90%) and in
their amino acid sequences (95%). The amino acid sequences of Adh1p and Adh2p had only 22 differences out
of 347 residues, with no differences in the groups directly
involved in catalysis (Ganzhorn et al., 1987).
ADH3 (YMR083W) encoding Adh3p

The presence of Adh3p in respiratory-deficient mutants
proved that nuclear DNA encodes the synthesis of this
mitochondrially bound enzyme (Wiesenfeld et al., 1975).
Young & Pilgrim (1985) isolated and sequenced the ADH3
gene, and the nucleotide sequence indicated a 73% and 74%
identity with ADH1 and ADH2, respectively. Wiesenfeld
et al. (1975) assigned Adh3p its tetrameric structure and the
amino acid similarity of the predicted Adh3p polypeptide to
Adh1p and Adh2p was 79% and 80%, respectively. All the
active site, cofactor-binding and noncatalytic zinc-binding
residues identified in S. cerevisiae Adh1p by Jornvall (1977)
are conserved in both Adh2p and Adh3p.
The ORF-encoding Adh3p has a highly basic 27 amino
acid amino terminal extension relative to Adh1p and Adh2p.
Gene fusion showed that the amino terminus of Adh3p
contained the information for targeting the protein to, and
transporting it into the mitochondrion (van Loon & Young,
1986). The amino acid sequence and secondary structure(s)
of the leader sequence, as well as the adjacent sequences in
the mature amino terminus of Adh3p, all contribute information for normal mitochondrial binding, importing and
processing (Mooney et al., 1990).
ADH4 (YGL256W) encoding Adh4p

ADH4 is the most distal marker on the left arm of chromosome VII and both restriction and genetic analysis of the
chromosome copy of ADH4 indicated that it was situated
near a telomere (Walton et al., 1986). Paquin & Williamson
(1986) described ADH4 as a new ADH isozyme, or that it
regulated the expression of an ADH. Sequencing analysis of
ADH4 revealed a long ORF that was not homologous to
other yeast ADHs and only distantly related to other
characterized eukaryotic ADHs (Paquin & Williamson,
1986). Analysis of the sequence of the hypothetical Adh4p
protein showed that it did not contain structurally or
functionally important amino acid residues that were conserved between yeast Adh1p and horse liver ADH. The
hypothetical ADH4 gene product did, however, show a
strong homology to the iron-activated ADH from the
bacterium Zymomonas mobilis. This homology suggested
that ADH4 did encode an ADH, but one distinct from any
other that has been described in eukaryotes (Williamson &
Paquin, 1987).
The presence of a TATAA sequence upstream of the
transcription start and the moderate codon bias suggested
that it may be a functional yeast gene. Its location near the
end of chromosome VII is interesting in view of the fact that
many of the genes located at the ends of chromosomes in S.
cerevisiae encode enzymes involved in fermentation and
glycolysis (Mortimer & Schild, 1985). Unlike Adh1p, Adh2p
and Adh3p, which are thought to function as tetramers
(Leskovac et al., 2002), Adh4p is a dimeric protein that
normally occurs in low concentrations in laboratory strains
(Drewke & Ciriacy, 1988). Contrary to the suggestion of
Williamson & Paquin (1987), Adh4p is activated by zinc
ions, like the other yeast ADH isozymes, and not by ferrous
ions as is the case with the structurally similar ADH from Z.
mobilis (Drewke & Ciriacy, 1988).
ADH5 (YBR145W) encoding Adh5p

ADH5 was first identified through sequencing of chromosome II (Feldmann et al., 1994) and shares a 76%, 77%, and
70% sequence identity with ADH1, ADH2 and ADH3,
respectively (Feldmann et al., 1994; Ladriere et al., 2000).
No information is currently available regarding characterization of the putative ADH5 gene product.
ADH6 (YMR218C) encoding Adh6p

The ADH6 (Gonzalez et al., 2000) gene product was the first
NADPH-dependent medium-chain ADH to be characterized in S. cerevisiae (Larroy et al., 2002b). It is also the first
cinammyl ADH and member of the MDR superfamily
whose three-dimensional crystal structure has been determined (Valencia et al., 2004). The heterodimeric enzyme
Adh6p consists of two 40 kDa subunits: one in the apo
conformation and the second in the holo conformation
(Valencia et al., 2004). Adh6p exhibits conservation of the
zinc-signature, as well as amino acid sequences in the
substrate and coenzyme-binding domains characteristic of
the zinc-containing MDR enzymes (Gonzalez et al., 2000;
Larroy et al., 2002b). Adh6p showed only a 26% sequence
identity to the tetrameric enzyme family (Valencia et al.,
2003) and exhibited a strict specificity for NADPH (Larroy
et al., 2002b).

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970
ADH7 (YCR105W) encoding Adh7p

Adh7p has a 64% sequence identity with Adh6p and the
purified ADH7 gene product is a homodimer whose reductase
activity is about fivefold that of the dehydrogenase activity
(Larroy et al., 2002a, b). The phylogenetic tree constructed
from the MDRs identified in the genomes of S. cerevisiae,
Escherichia coli, Drosophila melanogaster and Caenorhabditis
elegans placed Adh7p in a family of enzymes structurally
related to cinnamyl ADHs (Larroy et al., 2002a, b).
Tertiary and quaternary structure

Active centres
Relationships known from tertiary structures of dehydrogenases show that the constituent monomers are separated
into two domains, namely a coenzyme-binding domain
and a catalytic domain. The three-dimensional structure of
the active site of the S. cerevisiae enzyme revealed the
presence of a hydrogen-bonded proton relay system (Leskovac et al., 1998). On the basis that each individual chain
contains one reactive sulphydryl group and, by binding one
atom of zinc and 1 mol of NAD1/NADP1, it is capable of
forming an independent active centre within the quaternary
structure of the active tetramer (Harris, 1964). Adh1p has a
methionine residue at position 294 (numbered as in the horse
liver enzyme), whereas isozymes Adh2p and Adh3p have
leucine. Apart from these differences, the active sites of the S.
cerevisiae enzymes are the same (Ganzhorn et al., 1987). It has
been proposed that the shape or the accessibility of the
catalytic pocket appears to be different in the yeast and horse
liver enzymes and that it is possible to alter the specificity of
the enzyme without sacrificing catalytic power (Green et al.,
1993). Such approaches are limited by the lack of data on the
tertiary and quaternary structure of tetrameric S. cerevisiae
ADH. Crystallization of Adh1p has been reported repeatedly,
but the crystals are seemingly not very useful in X-ray
diffraction studies (Ciriacy, 1997). The size and shape of the
Adh6p active site appears to be adapted to the bulky and
hydrophobic substrates of cinammyl ADHs. The crystal
structure of this enzyme showed that its specificity towards
NADP(H) is achieved mainly by tripod-like interactions of the
cofactor terminal phosphate group with certain side chains
(Valencia et al., 2004).
Coenzymes and metal-binding sites

All MDR enzymes utilize NAD(H) or NADP(H) as a
cofactor and have one zinc ion with a catalytic function at
the active site (Branden et al., 1975). Early studies showed
that the active enzyme binds 4 mol of NAD1 (Karlovic et al.,
1976) and four atoms of zinc. Karlovic et al. (1976) also
demonstrated that binding of the coenzymes was linear over

Journal compilation
O. de Smidt et al.
a wide temperature range, both at the level of binary and

ternary complexes, and thermodynamic parameters showed
no close similarity between heat and entropy changes
associated with NAD and NADH binding. Zinc atoms are
essential for maintaining the quaternary structure of the
enzyme and both zinc and the coenzyme are bound at, or
near to, each of the four reactive cysteines (Harris, 1964).
Zinc is one of the principal trace elements in biology, with
structural or enzymatic roles in hundreds of proteins (Vallee
& Falchuk, 1993). Saccharomyces cerevisiae Adh1p, Adh2p
and Adh3p contain one catalytic zinc atom and a second
zinc atom, which plays a prominent conformational role,
probably through stabilization of the tertiary structure. The
second zinc is located at the periphery of the molecule and
the external localization of this structural zinc affects local
conformations of the enzyme (Magonet et al., 1992). The
different coenzyme-binding domains have extensive similarities, are composed of two mononucleotide-binding units
and resemble the tertiary structures of kinases and some
other proteins (Rossman et al., 1975)
The important role of the zinc atom in alcohol oxidation
is to stabilize the alcoholate ion for the hydride transfer step
in the reverse direction. Zinc functions as an electron
attractor, which gives rise to an increased electrophilic
character of the aldehyde, consequently facilitating the
transfer of a hydride ion to the aldehyde. Thus, the proposed
mechanism is essentially electrophilic catalysis mediated by
the active site zinc atom (Leskovac et al., 2002).
Kinetic characteristics
The first biochemical data on Adh1p and Adh2p showed
that the kinetic properties of both enzymes favoured alcohol
production. Under the conditions of a high ethanol concentration and the efficient removal of acetaldehyde, both
enzymes could function in the oxidation of ethanol (Heick
et al., 1969). Wills (1976) later stated that Adh1p was
normally constitutive under laboratory conditions, had a
high Km value for ethanol (17 00020 000 mmol L 1) (Thomson et al., 2005) and, therefore, seemed chiefly responsible
for the production of ethanol during anaerobic growth. If
levels of intracellular ethanol were low, Adh2p would
produce acetaldehyde and NADH at a faster rate than Adh1p
(Wills et al., 1982). Kinetic investigation of commercially
available ADH showed it to be capable of oxidizing all
primary alcohols with chain lengths of between two and 10
carbon atoms (Schopp & Aurich, 1976), and the activity of
Adh1p decreased with increasing chain length of the primary alcohols (Ganzhorn et al., 1987). The substrate specificity of Adh1p is restricted to primary unbranched aliphatic
alcohols and any branching decreased the activity and
efficiency of the enzyme (Leskovac et al., 2002). Cyclic
alcohols (benzyl alcohol, cyclohexanol) were not oxidized
971
in detectable amounts (Drewke & Ciriacy, 1988) and thiol

compounds exerted no effect on this isozyme (Cheng & Lek,
1992). It was also reported that overexpressed Adh1p
reduced formaldehyde (FA) to methanol in vivo (Grey
et al., 1996) and was able to provide a considerable degree
of protection against cadmium (Yu et al., 1991).
Adh2p has a low Km for ethanol (600800 mmol L 1) and
is found only in aerobically grown yeast cells (Wills, 1976;
Thomson et al., 2005). These findings concur with its role as
a major ethanol oxidizer. For all alcohols, normalized
reaction rates with Adh2p were about threefold faster than
with Adh1p (Leskovac et al., 2002). Some contradiction is
found in the literature regarding the kinetic characteristics
of Adh1p and Adh2p. Some reports state remarkably similar
normalized reaction activities for both Adh1p and Adh2p
under conditions of low substrate concentration, even
though the kinetic characteristics of the enzymes are very
different (Dickinson & Dack, 2001).
The mitochondrial enzyme Adh3p (Bakker et al., 2000)
showed great affinity for alcohols with double bonds conjugated to the alcohol function (Wiesenfeld et al., 1975). The
methionine (Adh1p) or leucine (Adh2p and Adh3p) at
position 294 by itself had no interaction with ethanol or
propanol (Ganzhorn et al., 1987).
Adh4p has different substrate specificity and pH profiles
compared with other ADH isozymes (Drewke & Ciriacy,
1988). Although Adh2p and Adh4p differ remarkably in
almost all the kinetic parameters (Lutstorf & Megnet, 1968),
the latter resembles Adh1p in its kinetic constants. Adh4p is
seemingly more specific, however, because only ethanol and
n-propanol are oxidized, whereas isomers of aliphatic alcohols, secondary alcohols and glycerol are not used (Drewke
& Ciriacy, 1988). No data on the kinetic characteristics of
Adh5p are presently available.
Adh6p accepts a wide range of compounds as substrates,
including linear and branched-chain primary alcohols and
aldehydes, substituted cinnamyl alcohols and aldehydes as well
as substituted benzaldehydes and their corresponding alcohols. It is able to produce 2,3-butanediol from acetoin during
fermentation (Gonzalez et al., 2000; Larroy et al., 2002a, b).
In general, the substrate specificity of Adh7p is quite
similar to that of Adh6p (Larroy et al., 2002a). It showed the
same activity towards linear and branched-chain alcohols,
but much higher catalytic efficiencies towards the oxidation
of cinnamyl alcohols and aliphatic alcohols (Larroy et al.,
2002b).
ing proof of a controlling site involved in carbon catabolite

repression in a eukaryote (Ciriacy, 1975b, 1976). The first
gene associated with this function was ADR1, a positive
regulatory gene specifically activating the expression of the
structural gene ADH2 under derepressed conditions (Ciriacy, 1979). ADR1 encodes the trans acting protein (Adr1p)
containing two zinc fingers and an adjacent region on the
amino-terminus side, which together are essential for DNA
binding (Blumberg et al., 1987). Initiation of transcription
from most known eukaryotic promoters is positively regulated by the binding of specific transcriptional activator
proteins to the enhancer region of the upstream activation
sequence (UAS) of a promoter (Hope & Struhl, 1985).
Two unusual features upstream of the ADH2 promoter, a
22-bp perfect dyad sequence and a (dA)20 tract, were
identified (Russell et al., 1983a). The ADH2 promoter may
normally be in an inactive conformation in the yeast
chromosome and derepression requires positive activation
by Adr1p that is mediated through the 22-bp perfect dyad
(UAS1) (Beier et al., 1985). The Adr1p monomers are able to
form one of two complexes: complex I corresponds to the
binding of one molecule to the cis acting element UAS1 and
complex II corresponds to the binding of two molecules to
UAS1 (Thukral et al., 1991). A GC rich, 20 bp sequence
(UAS2) upstream of UAS1 was identified and proposed to
act synergistically as a binding site for a protein that
interacts with Adr1p to activate the expression of ADH2
(Yu et al., 1989). In vitro binding data to this cis-acting
element suggest that Adr1p binds with a low affinity to
UAS2, most likely at the AGGAGA sequence. Other interpretations, such as an indirect effect or a direct protein
protein interaction, are also possible but seem less likely
(Donoviel et al., 1995).
Synthesis of Adr1p is 1016-fold greater during growth
on ethanol than during growth on glucose. This derepression of ADR1 protein translation was found to occur within
4060 min of glucose depletion. Glucose, therefore, represses ADH2 expression by considerably decreasing the
rate of Adr1p synthesis. Cook & Denis (1993) established
that a 510 bp untranslated leader sequence of the ADR1
mRNA played a role in the increased rate of ADR1 mRNA
degradation under conditions of growth on glucose as
compared with growth on ethanol. Other positive factors
influencing ADH2 expression were also proposed, because
excess Adr1p could not overcome a three- to fourfold
inhibition in ADH2 transcription caused by multiple promoters on a multicopy vector (Irani et al., 1987).
Regulation
Regulation of ADH2 mediated by Adr1p
Other elements participating in ADH2

regulation
The earliest study of regulation of the ADH genes was

documented in the mid-1970s and provided the first defin-
Genetic and biochemical analysis showed that expression of

the Adh2p structural gene was under the control of at least

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972
O. de Smidt et al.
24 other unlinked genetic elements or proteins, most of

which influence ADH2 expression mainly in a direct fashion
(Table 1). Several of the genes appear likely to do so through
control of Adr1p, whether by mRNA translation, phosphorylation or protein interaction. In contrast to the situation
with most genes that are subject to catabolite repression,
there is no evidence for an MIG1-binding site in the ADH2
promoter (Denis & Audino, 1991).
Influence of chromatin remodelling on ADH2

expression
Yeasts have to respond very rapidly to environmental
changes. Therefore, their chromatin is maintained in a state
of transcriptional readiness, even when the cells are transcriptionally inactive. The ADH system serves as an ideal
model to detect localized differences in chromatin structure,
Table 1. Elements other than Adr1 involved in the regulation of the ADH2 structural gene
Element
Postulated mode of action
References
ADR3
CCR1
Cis-acting regulatory locus required for glucose regulation of the ADH2 gene
Encodes a constitutive protein that acts, probably posttranscriptional, in concert with or through
Adr1p
Regarded as a negative regulatory factor because of its complete recessiveness. It is not yet
established whether ADR4 acts specifically on the ADR1 or ADH2 gene or whether its gene product
constitutes a regulatory element with a pleiotropic action spectrum
Contains an ORF encoding a protein that has a potential metal-binding finger domain. It appears to
require an ADH2 sequence located downstream to or including the TATAA box and may act
subsequent to Adr1p, but before translation of ADH2 mRNA
Affect transcription independently of the upstream regulatory sequences. They are not specific to
the regulation of carbon metabolism and function as general effectors of transcriptional processes
Also affects transcription independently of the upstream regulatory sequences. Ccr4p contains two
distinct glutamine and leucine rich regions that presumably play an important role in protein
interactions that mediate the regulatory role of Adh2p
Encode a group of factors involved in repressing the transcription of HIS3 from a noncanonical
TATA. MS and two hybrid analyses identified Not1-Not5 as proteins associated with the Ccr4p
complex, implying that it should also be positively involved in gene transcription
Encode proteins that coimmunoprecipitate the Ccr4p and Not proteins. Defects in these Srb
proteins affect expression at the ADH2 locus in a manner similar to that observed for defects in
Ccr4p complex components
Although cAPK inactivates ADH2 expression by inhibiting Adr1p function through phosphorylation,
either directly or of another protein required for Adr1p activity, it does not appear responsible for
the glucose to ethanol transition in controlling ADH2 expression
Appears to activate ADH2 expression by turning off Adr1p function independently of cAPK. SCH9
derepression does not operate exclusively through Adr1p. Because Sch9p does not act through
UAS1, it may control factors that act through other activation sequences or through factors
controlling the general transcriptional machinery
Are trans-acting elements unlinked to Adr1p that affect ADH2 expression under both repressing
and derepressing growth conditions. The gene products allow ADH2 expression to efficiently
derepress in the absence of Adr1p as do cre1 and cre2
Required for maintaining high levels of ADR1 RNA when growing on glucose, but have little effect
during growth on ethanol. Whereas the physiological role of the SAF genes is unclear, they appear
to be expressed with both ethanol and glucose as carbon sources and no direct evidence exists that
they influence genes other than ADR1 and ADH2
Encodes a nuclearly localized protein whose expression is not regulated by glucose availability. REG1
is the first negative genetic element identified that affects ADH2 expression through an ADR1
dependent pathway via an additional step in transcriptional activation that does not involve the
phosphorylationdephosphorylation of Adr1p
Encodes the regulatory subunit of cAPK and influences ADH2 expression in an Adr1p-dependent
manner
Has an essential role during the adaptation of yeast on ethanol by controlling the induction of many
genes in response to glucose depletion. It contributes to transcriptional derepression of ADH2
synergistically with the Adr1p dependent UAS1 element
Strains defective in this gene allow SNF1 dependent constitutive activation of ADH2 expression by
Adr1
Beier & Young (1982)

Denis & Gallo (1986)
ADR4
ADR6
CRE1 & 2
CCR4
NOT1-4
SRB9-11
cAPK
Sch9
ADR7-9
SAF1-3
REG1
BCY1
Cat8
MoT1

Journal compilation
Ciriacy (1979), Denis et al.

(1981)
Taguchi & Young (1987),
OHara et al. (1988)
Denis (1984), Denis &
Malvar (1990)
Malvar et al. (1992)
Collart & Struhl (1994)
Liu et al. (2001)
Denis et al. (1992)
Denis & Audino (1991)
Karnitz et al. (1992)
Cook & Denis (1993)
Niederacher & Entian

(1991)
Donoviel et al. (1995)

Haurie et al. (2001),
Walther & Schuller (2001),
Tachibana et al. (2005)
Voronkova et al. (2006)
973
which can reflect changes in transcriptional activity. The

possibility exists that a decrease in transcriptional activity
changes the structure of chromatin in the whole ADH2
region, for instance, by a change of nucleosome spacing
(Sledziewski & Young, 1982). Under repressing conditions,
UAS1 and UAS2 are a nucleosome-free region, the TATA box
and RNA initiation sequence (RIS) is protected by nucleosomes
1 and 11 and a 20 bp poly (dA dT) tract is
included in the DNA wrapped around nucleosome
1.
Nucleosome mapping data show that glucose exerts its
inhibitory effect by keeping the relevant promoter sequences
(TATA box and RIS) in a nucleosomal configuration, thus
precluding their engagement with the transcription machinery (Verdone et al., 1997).
Chromatin remodelling that occurs at the S. cerevisiae
ADH2 promoter upon derepression is characterized by two
distinct structural alterations. The existence of two steps in
the process of chromatin remodelling suggests that at least
two functions can be attributed to Adr1p. First, the protein
reconfigures nucleosomes in the immediate vicinity of its
binding site, allowing the basal promoter elements to
assume the most appropriate structure for the subsequent
activation. Second, the protein recruits the transcription
machinery through its activation domain, allowing mRNA
accumulation (Di Mauro et al., 2000).
Three possible states of the ADH2 promoter have been
proposed: structurally and functionally inactive, structurally
derepressed but functionally inactive and fully derepressed
and functionally active. The three possible states of the
promoter, due to the absence or the presence of different
Adr1p portions, can be considered to be an ordered
sequence of events occurring at the ADH2 locus during
derepression (Di Mauro et al., 2000).
Verdone et al. (2002) analysed the in vivo chromatin
structure and the kinetics of transcriptional activation of
the S. cerevisiae ADH2 promoter as a function of genetically
modified histone acetylation levels. By genetically altering
the steady-state pattern of histone acetylation at the repressed ADH2 promoter, the structure of the nucleosome
containing the TATA box is destabilized, the promoter
becomes accessible to Adr1p and, when the cells are shifted
to derepressing conditions, the kinetics of mRNA accumulation is faster. Histone deacetylation/acetylation is, therefore,
directly involved in altering the chromatin structure at the
ADH2 promoter, influencing the binding of the major
transcriptional activator, with a concomitant effect on the
kinetics of mRNA accumulation.
Xella et al. (2006) later established the potential requirement for the chromatin remodelling factors Isw1p,
Isw2p and Chd1p in the regulation of the ADH2 gene.
They concluded that these factors contributed to the kinetics
of activation of ADH2 in response to glucose depletion
and were crucial in establishing the correct chromatin
structure across the ADH2-coding region, but seemed

largely dispensable for nucleosome organization at the
promoter.
Regulation of ADH1 , ADH3 , ADH4 and ADH5

Initially, it was stated that regulatory genes affecting Adh2p
mRNA expression appeared to have no effect on ADH1
expression (Denis et al., 1983). It was later established that
deletion of the genes CCR4 and CAF1 not only influenced
the repression of ADH2, but brought about a phenotype
with enhanced expression of ADH1 (Liu et al., 2001). A
conserved sequence UASRPG, present in various glycolytic
genes and ribosomal protein genes, also exists in the upstream region of ADH1 and appears to be essential for
efficient transcription (Santangelo & Tornow, 1990). However, this seems to be true only for cells in the exponential
growth phase, because the efficiency of the promoter is
virtually indistinguishable with or without the UASRPG
during growth on ethanol. In glucose-grown cells, the region
between
700 and
412 bp containing the UASRPG is
required for the start of ADH1 promoter activity during the
early exponential growth phase. The promoter region 750 bp
upstream of the ADH1 promoter features the presence of an
Adr1p-binding site. It is, therefore, tempting to speculate
that the downregulation of the long ADH1 promoter is
indeed due to activation of the upstream promoter by
Adr1p. Thus, it is possible that the presence of ethanol in
the culture broth rather than the absence of glucose
decreased the activity of the long ADH1 promoter in
glucose-grown cells (Ruohonen et al., 1995). Bird et al.
(2006) demonstrated that during zinc starvation, Zap1p
was required for the repression of ADH1 expression. Zap1p
binds upstream of the activator Rap1p and induces an
intergenic RNA transcript (ZRR1). ZRR1 expression leads
to the transient displacement of Rap1p and Gcr1p from the
promoter, resulting in ADH1 repression.
The complex regulation of ADH2 has made it an attractive model system for the genetic dissection of its transcriptional control, while the regulation of the remainder of the
ADHs has not received as much attention. Scant data are
available regarding the up- or downregulation of these genes
and no evidence exists regarding genetic elements associated
with this action. Similar to the behaviour of enzymes of the
tricarboxylic acid cycle (Heick et al., 1969), ADH3 is
repressed by glucose, although its repression is not as severe
as that of ADH2 (Ciriacy, 1975a; Young & Pilgrim, 1985),
and ADH3 is derepresssed after glucose is depleted from the
medium. Repression of ADH3 through expression of an
intergenic transcript under conditions of zinc starvation will
likely occur by a mechanism similar to that of ADH1, a
mechanism that may act to conserve zinc during a limitation
of this nutrient (Bird et al., 2006).

Journal compilation
974
ADH4 expression is upregulated by lithium, a compound

that is toxic to yeast cells grown on galactose, but is downregulated by dimethyl sulphoxide (DMSO) (Bro et al., 2003;
Zhang et al., 2003). Under conditions where Adh1p is
nonfunctional, spontaneous chromosomal amplification of
ADH4 was able to rescue the mutant phenotype (Dorsey
et al., 1993). ADH4 was also stringently regulated by zinc
without an observable phenotype (Yuan, 2000). ADH5
expression was unaffected by DMSO (Zhang et al., 2003)
and its transcription was significantly increased in an S.
cerevisiae mutant strain able to grow anaerobically on Dxylose, a carbon source not normally utilized by yeasts in the
absence of oxygen (Sonderegger et al., 2004).
Verified and probable functions

Kinetically, Adh1p seems to be chiefly responsible for the
production of ethanol from acetaldehyde in cells grown
anaerobically or in the presence of a glucose excess, whereas
Adh2p primarily functions to convert ethanol accumulated
during aerobic growth to acetaldehyde, with the concomitant
reduction of NAD1. Adh1p can also accomplish this task,
though presumably less efficiently. These two enzymes contribute to the economy of the cell by stabilizing the NAD1
NADH ratio. In the case of these two isozymes the possibility
of inter-substitution seems to exist; for instance, cells lacking
Adh2p activity can grow on ethanol as a carbon source under
aerobic conditions (Wills, 1976; Wills et al., 1982).
Wenger & Bernofsky (1971) reported that mitochondrial
Adh3p was responsible in part for the coupled respiration of
yeast mitochondria with ethanol as a substrate. Disruption
mutants of ADH3 showed no discernible mutant phenotype
other than the lack of Adh3p activity (Young & Pilgrim,
1985). Bakker et al. (2000) demonstrated that Adh3p was
involved in the shuttling of mitochondrial NADH to the
cytosol, where it is deoxidized by the external NADH
dehydrogenases. Their results supported the hypothesis that
Adh3p forms part of the ethanolacetaldehyde shuttle that is
necessary for the reoxidation of mitochondrial NADH
under anaerobic conditions.
The Adh4p protein has been characterized as a zinc-dependent ADH that was thought to be minimally expressed, if at all
(Drewke & Ciriacy, 1988). Williamson & Paquin (1987) were
unable to detect ADH4 transcripts on Northern blots of
laboratory strains grown on glucose or ethanol. Activity was
only observed upon insertion of a Ty transposon at ADH4 or
amplification of ADH4. It may be a cryptic gene with no
function or simply a gene that is not necessary under laboratory
conditions. Genetic and physiological analysis showed that
disruption of ADH4 did not influence the viability of the yeast
cell, nor was the enzyme responsible for the decrease in ethanol
production in adh1adh4 quadruple deletion mutants (Drewke
et al., 1990). It was suggested recently that ADH4 encoded the

Journal compilation
O. de Smidt et al.
major cytosolic ADH in the two brewing yeast strains 2DGR19

and NCYC1245 in which elevated ADH4 mRNA levels could be
detected under conditions, of high ethanol production (Mizuno et al., 2006). Yuan (2000) hypothesized that the induction of
ADH4 expression under low-zinc conditions suggested that
under these conditions the protein functioned as a back-up for
Adh1p. Bird et al. (2006) later also demonstrated the same
regulation pattern during zinc starvation and suggested two
possible models for induction: as Adh4p binds less zinc per
subunit than Adh1p, the Adh4p enzyme could be a more
efficient ADH during zinc limitation or Adh4p might bind iron
when zinc levels are limiting.
Drewke et al. (1990) found that a deletion mutant strain
lacking adh1 to adh4 was still able to produce ethanol when
grown on glucose as a carbon source. It is, therefore,
reasonable to believe that in this case Adh5p might have
been the enzyme capable of producing ethanol from acetaldehyde.
A search was undertaken to discover the genes and
enzymes used by S. cerevisiae in the catabolism of leucine
to isoamyl alcohol (Dickinson et al., 1997), valine to
isobutanol (Dickinson et al., 1998) and isoleucine to active
amyl alcohol (Dickinson et al., 2000). As long as the yeast
had one functional enzyme out of Adh1pAdh5p or Sfa1p, it
was viable and any one of these six enzymes was sufficient
for the final stage of amino acid catabolism, namely the
conversion of an aldehyde to a long chain or a complex
alcohol (Dickinson et al., 2003).
The specificity of the substrate and cofactor strongly
supports the physiological involvement of Adh6p in aldehyde reduction rather than in alcohol oxidation and under
oxidative conditions allows the yeast to use 2,3-butanediol
as a carbon and energy source (Larroy et al., 2002a). The
potential role of Adh6p in S. cerevisiae is not easy to
ascertain when simply considering its structural similarity
to plant cinnamyl ADHs. It may afford the yeast the capacity
to live in ligninolytic environments where products derived
from lignin biodegradation may be available. Another
potential function may include the biosynthesis of fusel
alcohols (Larroy et al., 2002a) and most certainly NADP(H)
homeostasis (Larroy et al., 2003). Recently, genome-wide
transcription analysis also identified the ADH6 gene as
encoding NADPH-dependent 5-hydroxymethyl furfural
(HMF) reduction activity (Petersson et al., 2006). It is also
plausible that manipulation of the levels of Adh6p and
Adh7p could be used by the fermentation industry to alter
the organoleptic properties of fermented beverages (Larroy
et al., 2002b).
Outlook
Even though the ADHs of S. cerevisiae have been intensively
investigated, many aspects remain that warrant further
research. The possibility of functional substitution among

the different enzymes remains an interesting concept.
Further data on this would contribute to assessing the in
vivo roles of the enzymes. These investigations should be
conducted not only under standard growth conditions or
conditions of carbon repression, but also under conditions
of other nutrient limitations such as a zinc limitation.
Biochemical analysis of Adh5p has not yet received much
attention. Information on Adh5p could provide an insight
regarding the function of this enzyme in, for instance, amino
acid metabolism. It is also appealing to consider the
prospect of chimeric enzymes with improved or dual functions.
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The Alcohol Dehydrogenases of Saccharomyces Cerevisiae - A Comprehensive Review

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MINIREVIEW

The alcohol dehydrogenases of Saccharomyces cerevisiae : a

Correspondence: Jacobus Albertyn,

FEMS Yeast Res 8 (2008) 967978

easy availability of yeast cells. Physiologically, the ADH

r 2008 University of the Free State

overview regarding the primary and secondary structure,

Classification of the ADHs

while studying the mechanisms of mitochondrial oxidation

Primary and secondary structure

r 2008 University of the Free State

ADH1 (YOL086C) encoding Adh1p

FEMS Yeast Res 8 (2008) 967978

Alcohol dehydrogenases of Saccharomyces cerevisiae

enzyme. It is also possible that Adh1p binds Mg21 in vivo

ADH2 (YMR303C) encoding Adh2p

ADH3 (YMR083W) encoding Adh3p

ADH4 (YGL256W) encoding Adh4p

FEMS Yeast Res 8 (2008) 967978

ADH5 (YBR145W) encoding Adh5p

ADH6 (YMR218C) encoding Adh6p

r 2008 University of the Free State

ADH7 (YCR105W) encoding Adh7p

Tertiary and quaternary structure

Coenzymes and metal-binding sites

r 2008 University of the Free State

a wide temperature range, both at the level of binary and

FEMS Yeast Res 8 (2008) 967978

Alcohol dehydrogenases of Saccharomyces cerevisiae

in detectable amounts (Drewke & Ciriacy, 1988) and thiol

ing proof of a controlling site involved in carbon catabolite

Other elements participating in ADH2

The earliest study of regulation of the ADH genes was

Genetic and biochemical analysis showed that expression of

FEMS Yeast Res 8 (2008) 967978

r 2008 University of the Free State

24 other unlinked genetic elements or proteins, most of

Influence of chromatin remodelling on ADH2

Postulated mode of action

Beier & Young (1982)

r 2008 University of the Free State

Ciriacy (1979), Denis et al.

Collart & Struhl (1994)

Liu et al. (2001)

Denis et al. (1992)

Denis & Audino (1991)

Karnitz et al. (1992)

Cook & Denis (1993)

Niederacher & Entian

Donoviel et al. (1995)

FEMS Yeast Res 8 (2008) 967978

Alcohol dehydrogenases of Saccharomyces cerevisiae

which can reflect changes in transcriptional activity. The

FEMS Yeast Res 8 (2008) 967978

structure across the ADH2-coding region, but seemed

Regulation of ADH1 , ADH3 , ADH4 and ADH5

r 2008 University of the Free State

ADH4 expression is upregulated by lithium, a compound

Verified and probable functions

r 2008 University of the Free State

major cytosolic ADH in the two brewing yeast strains 2DGR19

FEMS Yeast Res 8 (2008) 967978

Alcohol dehydrogenases of Saccharomyces cerevisiae

research. The possibility of functional substitution among