Báo cáo khoa học: Weak organic acid stress inhibits aromatic amino acid uptake by yeast, causing a strong influence of amino acid auxotrophies on the phenotypes of membrane transporter mutants ppt

Weak organic acid stress inhibits aromatic amino acid uptake by yeast,
causing a strong influence of amino acid auxotrophies on the
phenotypes of membrane transporter mutants
Bettina E. Bauer
1,
*, Danielle Rossington
2,
*, Mehdi Mollapour
3,
*, Yasmine Mamnun
1
, Karl Kuchler
1
and Peter W. Piper
3
1
Department of Molecular Genetics, University and BioCenter of Vienna, Austria;
2
Unilever Research Colworth, Sharnbrook,
Bedford, UK;
3
Department of Biochemistry and Molecular Biology, University College London, UK
The ability of yeasts to grow in the presence of weak organic
acid preservatives is an important cause of food spoilage.
Many of the determinants of acetate resistance in Sac-
charomyces cerevisiae differ from the determinants of
resistance to the more lipophilic sorbate and benzoate.
Interestingly, we show in this study that hypersensitivity to
both acetate and sorbate results when the cells have auxo-
trophic requirements for aromatic amino acids. In trypto-
phan biosynthetic pathway mutants, this weak acid

hypersensitivity is suppressed by supplementing the medium
with high levels of tryptophan or, in the case of sorbate
sensitivity, by overexpressing the Tat2p high affinity tryp-
tophan permease. Weak acid stress therefore inhibits uptake
of aromatic amino acids from the medium. This allows
auxotrophic requirements for these amino acids to strongly
influence the resistance phenotypes of mutant strains. This
property must be taken into consideration when using these
phenotypes to attribute functional assignments to genes. We
show that the acetate sensitivity phenotype previously
ascribed to yeast mutants lacking the Pdr12p and Azr1p
plasma membrane transporters is an artefact arising from
the use of trp1 mutant strains. These transporters do not
confer resistance to high acetate levels and, in prototrophs,
their presence is actually detrimental for this resistance.
Keywords: Saccharomyces cerevisiae; weak organic acid food
preservatives; plasma membrane transporters; Pdr12p;
Azr1p.
The resistance of yeasts to the small number of weak
organic acids allowed in food preservation allows these
organisms to cause large-scale spoilage of preserved foods
and beverages [1]. Saccharomyces cerevisiae is able to grow
in the presence of sorbate due to the War1p transcription
factor-dependent induction of a single ATP-binding cassette
(ABC) transporter, Pdr12p [2,3]. So strong is this Pdr12p
induction in sorbate-stressed cells, that levels of this
transporter in the plasma membrane approach those of
the most abundant plasma membrane protein, plasma
membrane H
+

-ATPase [1,2].
Pdr12p appears to be acting as an efflux pump for weak
organic carboxylate anions. It has been directly shown that
it lowers the intracellular levels of benzoate and fluorescein
by catalysing an active efflux of these compounds from the
cell [2,4]. The inhibitory effects of different organic acids on
cells of the Dpdr12 mutant indicate that Pdr12p confers
resistance to sorbate, benzoate and aliphatic short chain
(C
3)8
) carboxylic acids of reasonable water solubility [5,6].
A similar spectum of acids is also capable of mediating the
War1p-dependent induction of Pdr12p [3,6]. These are acids
that cannot generally be degraded by S. cerevisiae, instead
exerting appreciably cytotoxic or cytostatic effects mainly
through their ability to disrupt membrane structures [1].
Pdr12p does not confer resistance to either acetate (this
study) or highly lipophilic, long chain acids [5].
Acetate is far less inhibitory to yeasts than the more
lipophilic sorbate (trans,trans-hexanedienoate), even though
these are two carboxylate compounds of identical pKa
(4.76) [2,7–9]. This is thought to be because the latter
compound has a much higher capacity to dissolve in
membranes and so disorder membrane structure [1]. Nev-
ertheless S. cerevisiae is often inhibited by the presence of
high acetate levels in wine fermentations [10], partly through
the glyoxylate cycle enzymes needed for the assimilation of
acetate and propionate in aerobic cultures being glucose-
repressed ([11] and references therein). Our initial studies on
Dpdr12 and wild-type cells indicated the former mutant to

be more sensitive to high acetate levels [2]. A mutant lacking
another plasma membrane transporter, Azr1p, was also
shown to be acetate sensitive [12].
We show here that these effects on acetate resistance are
only seen with auxotrophic mutants that need to assimilate
aromatic amino acids from the culture medium. This
appears to have led to incorrect assignments of function to
the yeast Pdr12p and Azr1p transporters in studies that have
used trp mutant backgrounds. Our data indicate that certain
auxotrophic markers present in widely used yeast strains
Correspondence to P. W. Piper, Department of Biochemistry and
Molecular Biology, University College London, London WC1E 6BT.
Fax: + 44 207 6797193, Tel.: + 44 207 6792212,
E-mail:
Abbreviation: ABC, ATP-binding cassette.
Note: *The first three authors contributed equally to this work.
(Received 30 April 2003, revised 30 May 2003,
accepted 3 June 2003)
Eur. J. Biochem. 270, 3189–3195 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03701.x
may have led to other incorrect assignments of gene
function, in studies where these assignments are based on
drug susceptibility or resistance phenotypes.
Materials and methods
Yeast strains
The yeast strains used in this study are listed in Table 1. The
AZR1 gene was deleted using the kanMX4 cassette system
[13].
Construction of a
TAT2
overexpression plasmid

The TAT2 gene was amplified from genomic DNA by PCR,
using primers that introduced SalIsitesat)12 relative to the
ATG and at 162 bp downstream of the stop codon. This
PCR fragment was then Sal1-digested and cloned into the
SalI site located between the ADH1 promoter and termi-
nator of expression vector pAD4M [14]. The correct DNA
sequence of the TAT2 insert of the resulting vector was
confirmed by sequencing.
Yeast growth
Yeast was cultured aerobically at 30 °C in liquid YP
medium [1% (w/v) Difco Yeast Extract, 2% Bacto peptone,
20 mgÆL
)1
adenine], containing as carbon source either 2%
glucose (YPD); or 2% galactose (YPGalactose). Media were
titrated to pH 4.5 or pH 6.8 prior to autoclaving. Bioscreen
culture of strains on pH 4.5 YPD (30 °C) in the presence of
weak acid stress and the plating of strains on pH 4.5 YPD
plates containing stress agents were both as described
previously [2,4,5]. For Bioscreen culture cells were diluted in
fresh YPD, pH 4.5, and inoculated into the wells
of a Bioscreen microtitre plate (100 well honeycomb; Life
Sciences International) to give an inoculum size of
5.0 · 10
3
cellsÆml
)1
as described previously [5,15]. The stated
concentrations of acetic or sorbic acid were then added to
the wells. Growth at 30 °C with continuous shaking was

then monitored by change in 600 nm optical density using a
Labsystems Bioscreen automated turbidometric analyser
(Life Sciences International, Basingstoke, UK).
Analysis of Pdr12p levels
Pdr12p levels were analysed by immunoblotting, as des-
cribed earlier [2,3,6].
Results and discussion
Loss of Pdr12 or Azr1p only decreases acetate resistance
in strains with auxotrophic requirements for aromatic
amino acids
Our earlier studies of the growth of Dpdr12 and wild type
cells indicated that the former mutant is more sensitive to
acetate at pH 4.5 [2]. Another study reported that Azr1p, a
plasma membrane transporter of the major facilitator
superfamily, also confers acetate resistance [12]. We there-
fore constructed a double Dpdr12 Dazr1 mutant (Materials
and methods; Table 1), initially with the intention of
determining which of these proteins, Pdr12p or Azr1p,
might be more important for acetate resistance. We then
investigated the resistances of this double deletant and the
corresponding Dpdr12 and Dazr1 single gene deletes to
acetate and sorbate.
Growth on plates containing increasing levels of sorbate
or acetate indicated that the loss of Azr1p did not result in
increased sensitivity to either of these acids (Fig. 1; strains
with no auxotrophic requirements for aromatic amino
acids). It appeared though that acetate and sorbate sensi-
tivities were being enhanced with the loss of both Azr1p and
Pdr12p (Fig. 1, Dpdr12 Dazr1 mutant). Remarkably, the
loss of Pdr12p alone in this background resulted in slightly

increased resistance to acetate (Fig. 1).
To investigate the effects of high levels of Pdr12p
induction on acetate resistance we used a strain (Table 1,
Table 1. S. cerevisiae strains used in this study.
Strain Genotype Source
FY1679–11c MATa ura3-52 his3-D200 leu2-D1 Euroscarf
FY73 MATa ura3-52 his3-D200 Euroscarf
FY1679–28c MATa ura3-52 his3-D200 leu2-D1 trp1-D63 Euroscarf
YYM19 MATa ura3-52 his3-D200 leu2-D1 trp1-D63 Dpdr12::hisG [2]
FY809 MATa ura3-52 his3-D200 leu2-D1 Dpdr12::hisG FY1679–11c x YYM19
11c-azr1D MATa ura3-52 his3-D200 leu2-D1 Dazr1::kanMX4 This study
11c-pdr12D azr1D MATa ura3-52 his3-D200 leu2-D1 Dpdr12::hisG, Dazr1::kanMX4 This study
GAL1-PDR12 MATa ura3-52 his3-D200 leu2-D1 trp1-D63 PDR12::kanMX4-proGAL1 [6]
BY4741 MATa ura3-0 his3-D1 leu2-D0 met15-D0 Euroscarf
trp1D MATa ura3-0 his3-D1 leu2-D0 met15-D0 Dtrp1-kanMX4 Euroscarf
trp2D MATa ura3-0 his3-D1 leu2-D0 met15-D0 Dtrp2-kanMX4 Euroscarf
trp3D MATa ura3-0 his3-D1 leu2-D0 met15-D0 Dtrp3-kanMX4 Euroscarf
trp4D MATa ura3-0 his3-D1 leu2-D0 met15-D0 Dtrp4-kanMX4 Euroscarf
trp5D MATa ura3-0 his3-D1 leu2-D0 met15-D0 Dtrp5-kanMX4 Euroscarf
YPH499 MATa ura3-52 lys2-801
am
ade2-101
oc
trp1-D63 his3-D200 leu2-D1 [20]
YPH499 TRP
+
YPH499 trp1-D63::pRS304(TRP1) This study
war1-42 YPH499 war1-42 This study
3190 B. E. Bauer et al.(Eur. J. Biochem. 270) Ó FEBS 2003
GAL1-PDR12) in which the PDR12 gene is not under the

control of its normal promoter, but controlled instead by
the GAL1 promoter. This was because the PDR12 gene is
not normally induced by acetate [6], but during growth of
this GAL1-PDR12 strain on galactose can be induced to
levels similar to those seen in wild-type cells exposed to
sorbic acid stress [6]. Growth of normal yeast on galactose
relative to glucose slightly decreases acetate resistance
(Fig. 2), yet the strong Pdr12p induction with the growth
of the GAL1-PDR12 strain on galactose decreases acetate
resistance still further (Fig. 2). This contrasts with the
greatly increased sorbate resistance that results from same
GAL1 promoter-directed induction of Pdr12p [6]. It
provides yet further evidence that the induction of Pdr12p,
though beneficial for resistance to C
3)8
aliphatic carboxylic
acids and to sorbate [2,6], is actually somewhat detrimental
for resistance to acetate.
These results appeared to contradict our previous find-
ings of a decreased acetate resistance with the loss of Pdr12p
[2]. The strains used in these earlier studies were identical to
those used to obtain the data in Fig. 1, except that they
possessed the additional trp1-D63 mutation (Table 1).
When we retested the trp1-D63 strains used in our earlier
work [2], we found that they were generally less weak acid
resistant than the corresponding TRP
+
strains in Fig. 1.
Nevertheless, were still able to confirm our earlier findings
of a decreased acetate resistance with the loss of Pdr12p in

the trp1-D63 genetic background. We therefore investigated
Fig. 1. Growth of strains on YPD (pH 4.5) in the presence of the indicated levels of sorbate or acetate. Wild-type (W+), Dpdr12 (FY809) and Dazr1
single mutants, and a Dpdr12 Dazr1 double mutant (all isogenic to FY1679–11c; Table 1) were tested. An undiluted overnight culture, and 1 : 10
and 1 : 100 serial dilutions, were spotted onto solid pH 4.5 YPD and the plates photographed after 3 days at 30 °C.
Fig. 2. The effects of Pdr12p overexpression on growth in the presence of increased levels of acetate. The two trp1 strains analysed are isogenic but for
the PDR12 gene being under native promoter control in the wild-type (FY1679–28c; W+) and under GAL1 promoter control in the strain GAL1-
PDR12. An undiluted overnight culture, and 1 : 10 and 1 : 100 serial dilutions, were spotted onto solid pH 4.5 YPD or YPGalactose and the plates
photographed after 3 days at 30 °C.
Ó FEBS 2003 Auxotrophy effects on yeast transporter mutants (Eur. J. Biochem. 270) 3191
whether trp1-D63, and other mutations leading to require-
ments for aromatic amino acids, might be causing an
unusually high sensitivity to weak organic acid stress.
Auxotrophic requirements for aromatic amino acids
dramatically increase sensitivity to weak organic acid
stress, a sensitivity suppressed by amino acid
supplementation
Platings of an isogenic series of strains (haploids derived
from a YYM19 X FY73 cross; Table 1) on both sorbate-
and benzoate-containing agar confirmed that trp1-D63
enhances sensitivities to these two acids (Fig. 3). This was
apparent even in those cells that lacked Pdr12p (Fig. 3),
showing that trp1-D63 acts to increase weak acid sensitivity
independently of the Pdr12p ABC transporter. Similar
results (not shown) were obtained when these same strains
were plated in the presence of high concentrations of acetate.
Next, mutants were obtained from the Euroscarf collec-
tion that lack specific enzymes of the aromatic amino acid
biosynthetic pathway. These were then tested for their
sensitivities to both acetate and sorbate. Growth of the
wild-type (BY4741), together with its aro and trp mutant

derivatives on acetate and sorbate plates at pH 4.5 revealed
that the aro1D, aro2D, aro7D, trp1D, trp2D, trp3D, trp4D
and trp5D mutations all (in a similar manner to trp1-D63;
Fig. 3) hypersensitize cells to acetate and sorbate (not
shown). Defects in several of the enzymes of aromatic
amino acid biosynthesis therefore hypersensitize yeast to
weak organic acid stress.
We also investigated the growth of this same series of
strains after they had been inoculated into pH 4.5 liquid
medium, either with no weak acid present or with acetate
or sorbate (Materials and methods). Figure 4A,B shows
representative culture data for the TRP
+
strain BY4741
and its trp5D mutant derivative, either in the absence or the
presence of acetate stress. The trp5D mutant is clearly much
more sensitive than the wild-type to the inhibitory effects of
acetate under these conditions (Fig. 4A,B). The trp1D,
Fig. 3. Both the Dtrp1 and Dpdr12 mutations increase sensitivity to sorbate and benzoate. Isogenic haploid strains with the indicated abbreviated
genotypes (all of mating type a, and also ura3) were spotted (undiluted; also as 1 : 10 and 1 : 100 serial dilutions) onto pH 4.5 YPD containing the
indicated level of organic acid. The plates were photographed after 3d at 30 °C.
Fig. 4. Bioscreen culture of the TRP
+
BY4741 wild-type (A,C) and its
Dtrp5 mutant derivative (B,D), in the presence of no weak acid (j),
40m
M
acetate (n) and 80 m
M
acetate (m). Theculturesin(A)and(B)

were grown in the absence of any medium tryptophan supplement,
whereas the cultures in (C) and (D) were grown with a 50 m
M
tryp-
tophan supplementation.
3192 B. E. Bauer et al.(Eur. J. Biochem. 270) Ó FEBS 2003
trp2D, trp3D and trp4D mutants were also compromised
under these conditions (not shown), their growth being
essentially similar to that of the trp5D mutant cells shown in
Fig. 4.
All of these mutants, unlike the BY4741 parent, must
catalyse an uptake of tryptophan from the medium in order
to grow. Suspecting that it might be this tryptophan uptake
that is inhibited strongly by the weak acid stress, we
investigated the effects of supplementing the medium with a
high level of tryptophan (Fig. 4). While the trp5D mutant is
extremely acetate-sensitive, this hypersensitivity is almost
totally suppressed by a high level of tryptophan (Fig. 4B,D).
Such tryptophan supplementation could restore the growth
of the acetate-stressed trp5D mutant cells to that of control,
acetate-stressed wild-type (BY4741) cells lacking such
supplementation (Fig. 4A,D).
Liquid culture of strain BY4741 and its trpD mutant
derivatives in the absence or presence of sorbate showed
that trp mutants are also hypersensitized to sorbate stress
(consistent with the effects of the trp1-D63 mutation on
growth on pH 4.5 YPD agar containing sorbate; Fig. 3).
Figure 5 shows sample data for the TRP
+
BY4741 strain

and its trp5D mutant derivative grown in the presence of
0.9 m
M
or 1.8 m
M
sorbate. Again, sorbate hypersensitivity
was substantially suppressed with the addition of a high
level of tryptophan to the growth medium. The latter
supplementation almost restored the growth of the trp5D
mutant to that of sorbate-stressed wild-type cells lacking
such supplementation (Fig. 5). Control experiments showed
that these high tryptophan levels have no effect on Pdr12p
induction by sorbate (Fig. 6A). Furthermore, because
exogenous tryptophan suppresses both the acetate (Fig. 4)
and sorbate (Fig. 5B) sensitivities of trp mutant cells (only
the latter acid, not the former, being a Pdr12p inducer [6]) it
is clear that this suppression by exogenous tryptophan bears
no relationship to the weak acid inducibility of Pdr12p.
Overexpression of Tat2p increases sorbate resistance,
but only in
trp
mutant backgrounds
The above data reveals that a requirement for uptake of
aromatic amino acids from the culture medium leads to
unusually high sensitivity to weak organic acid stress. An
increased capacity for cells to catalyse uptake of aromatic
amino acids might therefore suppress this sensitivity. To
obtain evidence of whether this is the case, we studied the
effects of overexpressing the high affinity tryptophan perm-
ease, Tat2p [16]. The TAT2gene was placed under the control

of the strong, constitutive ADH1 promoter in the multicopy
vector pAD4M, thereby yielding the plasmid pTAT2
(Materials and methods). This construct and the empty
vector pAD4M were then introduced into both trp1-D63 and
TRP1 versions of strain YPH499 (Table 1). The increased
sorbate sensitivity due to trp1-D63 was substantially sup-
pressed by the pTAT2 overexpression vector, a plasmid
which could almost restore the growth of the sorbate-stressed
trp1-D63 cells to the level of growth displayed by an isogenic
TRP
+
prototroph (Fig. 7A). Furthermore, Pdr12p levels
were not affected by TAT2 overexpression (Fig. 6B),
indicating that this rescue of acid-stressed cells by pTAT2
acts independently of the Pdr12p transporter.
Fig. 5. Bioscreen culture of BY4741 wild-type (h, j) and Dtrp5 mutant
cells (m, n)atpH4.5.Cells were grown in the presence of either
0.9 m
M
(A) or 1.8 m
M
(B) sorbic acid, either in the absence (h, n)or
the presence (j, m) of tryptophan supplementation.
Fig. 6. Measurements of Pdr12p levels. (A) Both basal and sorbate-
induced levels of Pdr12p are not altered by a tryptophan supplemen-
tation (+). (B) Pdr12p levels do not undergo significant change in
Tat2p-overexpressing cells; also Tat2p overexpression in the war1-42
mutant does not restore Pdr12p induction.
Ó FEBS 2003 Auxotrophy effects on yeast transporter mutants (Eur. J. Biochem. 270) 3193
To corroborate the independence of the Tat2p effect from

Pdr12p function, we took advantage of a loss-of-function
mutant in War1p, the transcription factor responsible for
PDR12 induction upon acid stress challenge [3]. This
mutant, war1-42, fails to induce PDR12 expression when
challenged with sorbate and is therefore sorbate-hypersen-
sitive. We transformed this war1-42 mutant and its YPH499
parent (both trp1-D63 strains) with pTAT2 and the empty
pAD4M vector. Transformants were then tested for their
ability to grow in the presence of sorbate. While pAD4M
could not sustain the growth of the war1-42 mutant on
0.75 m
M
sorbate, pTAT2 could enable these cells to tolerate
sorbate at up to 1 m
M
(Fig. 7B). Hence, Tat2p overexpres-
sion can partially rescue the sorbate sensitivity of the war1-
42 mutant, a mutant lacking Pdr12p induction, in a trp1
genetic background.
Loss of Pdr12p or Azr1p increases acetate resistance
in prototrophic backgrounds
This investigation into the influences of aromatic amino
acid auxotrophy on weak acid resistance was initiated in
response to the finding that losses of Pdr12p or of Azr1p
appeared to be exerting opposite effects on acetate resistance
in trp1 and TRP
+
genetic backgrounds. In trp1 mutants the
losses of these transporters increase sensitivity to acetate
[2,12], whereas in TRP

+
cells the same losses are associated
with either neutral effects or a decreased sensitivity to
acetate (Fig. 1). Notably, neither Pdr12p nor Azr1p is
actually induced by acetate stress [6] (M. Mollapour,
unpublished results), although a GAL1 promoter-directed
induction of Pdr12p clearly results in a reduced resistance to
acetate (Fig. 2).
Bioscreen culture (Fig. 8) confirmed that the loss of
Azr1p alone does not increase the sorbate sensitivity of
TRP
+
cells, though this loss slightly increased the sorbate
sensitivity of the Dpdr12 mutant (Fig. 8B). Unexpectedly,
the Dpdr12 and Dazr1 single gene deletes grew considerably
better than the wild-type at pH 4.5 in the presence of
120 m
M
acetate (Fig. 8). After an extended lag phase, the
Dpdr12 Dazr1 double mutant also grew considerably better
than the wild-type (Fig. 8C). Loss of either Pdr12p or Azr1p
appears therefore to be beneficial for growth in the presence
of high concentrations of acetate. The reasons for this
improved growth are not yet clear, but one possibility is that
these mutants are not displaying the apoptotic events
normally seen in yeast cells treated with high levels of
acetate [17]. Such improved growth after an extended lag
phase was not indicated by the initial plating (Fig. 1),
underlining the importance of progressing from initial
plating assays (Figs 1–3) to detailed studies of growth

kinetics for any full characterization of the effects of a
mutation on growth in the presence of a stress agent
(Fig. 8). Importantly, this and other studies [18] strike a
note of caution, as they highlight the strong influences that
certain mutations in the genetic background can exert on
stress resistances. It is unlikely that Pdr12p or Azr1p confers
acetate resistance as suggested by earlier work [2,12]; indeed
their loss may actually elevate this resistance (Fig. 8).
Fig. 7. Effects of Tat2p overexpression on sorbate resistance. (A)
Overexpression of Tat2p increases sorbate resistance, but only in a trp
mutant background. (B) The same overexpression suppresses the
sorbate sensitivity of a mutant (war1-42) that is defective in Pdr12p
induction. Undiluted overnight cultures of the indicated transformants
were serially diluted, spotted onto pH 4.5 YPD, and the plates incu-
bated 3 days at 30 °C.
Fig. 8. Bioscreen culture of TRP
+
wild-type (j), Dpdr12 (r) and Dazr1
(m) single mutants and a Dpdr12 Dazr1 double mutant (s)atpH4.5.
Experiments were carried out in the presence of no stress agent (A),
0.9 m
M
sorbate (B), 80 m
M
acetate (C) or 120 m
M
acetate (D).
3194 B. E. Bauer et al.(Eur. J. Biochem. 270) Ó FEBS 2003
The bacterium Escherichia coli cannot synthesize aromatic
amino acids when it suffers severe oxidative stress. This

auxotrophy, the result of oxidation of the 1,2-dihydroxy-
ethyl thiamine pyrophosphate intermediate of transketolase,
is suppressed when cultures are supplemented with inter-
mediates (e.g. shikimate) that allow aromatic amino acid
synthesis to occur independently of the transketolase reac-
tion [19]. Weak acid stress in yeast is acting in a fundament-
ally different way. It is not generating an auxotrophy for
aromatic amino acids in wild-type cells, but rather is causing
high sensitivity to any requirement for the cells to catalyse
uptake of aromatic amino acids from the medium. Probably
this is due to the weak organic acid exerting a strong
inhibition of the activity of the Tat2p amino acid permease,
though this is not directly proven by this study.
Acknowledgements
This work was supported by project grants from the Biotechnology and
Biological Sciences Research Council (31/D17868 to PP) and the
Austrian Science Foundation (P-15934-B08 to KK); also in part by
funds from DSM Bakery Ingredients to PP and KK.
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Ó FEBS 2003 Auxotrophy effects on yeast transporter mutants (Eur. J. Biochem. 270) 3195