Dismutation of aldehydes catalyzed by alcohol dehydrogenases

Article abstract of DOI:10.1039/b001039l

The dismutation of aldehydes with the following three alcohol dehydrogenases, the mesophilic Saccharomyces cerevisiae ADH; the thermophilic Thermoanaerobium brockii ADH; and the recently isolated psychrophilic Moraxella sp. TAE123 ADH, was studied with high-resolution ¹H NMR spectroscopy. All three ADHs catalyzed the rapid dismutation of aldehydes to the corresponding alcohols and carboxylic acids.

Full text of DOI:10.1039/b001039l

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Dismutation of aldehydes catalyzed by alcohol dehydrogenases†
Kelly Velonia and Ioulia Smonou*
Department of Chemistry, University of Crete, Iraklio 71 409, Crete, Greece.
E-mail: smonou@chemistry.uch.gr
1
Received (in Cambridge, UK) 7th February 2000, Accepted 25th May 2000
Published on the Web 30th June 2000
The dismutation of aldehydes with the following three alcohol dehydrogenases, the mesophilic Saccharomyces
cerevisiae ADH; the thermophilic Thermoanaerobium brockii ADH; and the recently isolated psychrophilic
Moraxella sp. TAE123 ADH, was studied with high-resolution 1H NMR spectroscopy. All three ADHs catalyzed
the rapid dismutation of aldehydes to the corresponding alcohols and carboxylic acids.
The NADPϩ-dependent thermophilic alcohol dehydrogenase
Introduction
from Thermoanaerobium brockii (TBADH) is a medium-chain
zinc-containing enzyme,14,19 remarkably stable at temperatures
up to 85 ЊC. TBADH is also a type-A enzyme,20 which catalyzes
the hydride transfer generally to the Re face of carbonyl
compounds to afford the corresponding (S)-alcohols. TBADH
reacts mainly with secondary alcohols, acyclic and cyclic
ketones and primary alcohols.
Moraxella sp. TAE123 ADH is a psychrophilic alcohol
dehydrogenase. From its molecular characteristics it has been
classified in the longer-than-the-classical-type form of ADH
families.15 It has been characterized as a type-A enzyme.21
Moraxella sp. TAE123 ADH exhibits wide substrate specificity,
oxidizing mainly primary and secondary alcohols as well as
bulky aromatic alcohols (such as benzhydrol). From our previ-
ous studies we have found that it reduces butan-2-one to (S)-
butan-2-ol with high stereoselectivity.21
In this paper we demonstrate, by the use of high-resolution
1H NMR spectroscopy, that all the above three ADHs catalyze
the rapid dismutation of aldehydes to the corresponding alco-
hols and carboxylic acids. TBADH and Moraxella sp. TAE123
ADH represent the first examples of alcohol dehydrogenases
derived from extremophilic bacteria to exhibit dismutase activ-
ity. This fact reinforces the view that the dismutation reaction is
part of the physiological function of alcohol dehydrogenases.
Alcohol dehydrogenases (ADHs), obtained from a variety of
sources,1 are among the most extensively studied enzymes.
Their ability to catalyze the interconversion of alcohols to the
corresponding carbonyl compounds with high enantioselectiv-
ity has found numerous applications in asymmetric organic
synthesis.2 While this reaction has been considered to be their
physiological function,3 the oxidation of aldehydes to carb-
oxylic acids has mostly been unrecognized. For many years, the
dismutation of aldehydes into equimolar quantities of the
corresponding alcohols and carboxylic acids was thought to be
a unique, but insignificant, side reaction of horse liver ADH
(HLADH).4–6 This view was reinforced by the fact that
the alcohol dehydrogenase from Saccharomyces cerevisiae
(YADH), which is structurally and mechanistically similar to
HLADH, was reported to have no aldehyde dehydrogenase
activity.7 The detailed mechanism of aldehyde oxidation was
recently studied and was shown to be more complicated than
previously thought,8–10 involving the rapid dismutation of the
aldehyde prior to any production of NADH. Furthermore,
members of other major ADH classes, including Drosophila
melanogaster (DADH)11 and human liver ADH,12 have also
been reported to display aldehyde dehydrogenase function.
However, this reaction is still considered to be an intrinsic
characteristic of these enzymes, rather than a general property
of alcohol dehydrogenases.
In the present study we investigated systematically the dismu-
tation of aldehydes by using the following three representative
alcohol dehydrogenases: the mesophilic YADH,13 the thermo-
philic Thermoanaerobium brockii ADH (TBADH)14 and the
recently isolated psychrophilic Moraxella sp. TAE123 ADH.15
These enzymes are different in their temperature adaptation as
well as in their structural and molecular characteristics.
The mesophilic YADH belongs to the medium ADH family
and is a type-A enzyme transferring the pro-R hydrogen of C-4
of NADH to the Re face of carbonyl substrates and vice versa.16
It reduces a variety of aldehydes and oxidizes mainly primary
acyclic alcohols with stoichiometric consumption of NADH or
NADϩ respectively. Despite its in vivo role to reduce acetalde-
hyde to ethanol17 and its structural and mechanistic similarity
to HLADH, it was believed that YADH does not display dis-
mutase activity.7 Recent18 results, however, indicate that YADH
also catalyzes the dismutation of aldehydes into equimolar
quantities of the corresponding alcohols and carboxylic acids.
This result has been verified also by our experimental work.
Results and discussion
The ability of the selected dehydrogenases to catalyze the
dismutation of aldehydes was assayed with acetaldehyde and
propionaldehyde. According to the mechanism proposed by
Oppenheimer,8 dismutation proceeds without the net produc-
tion of NADH. The most appropriate method to follow these
spectrophotometrically silent reactions was 1H NMR spec-
troscopy. All reactions were carried out in 5 mm NMR tubes
and the spectra were acquired by using solvent (water) suppres-
sion with solvent presaturation. For acetaldehyde, the course of
the reaction was followed by integrating the methyl hydrogen
absorptions of all species present, i.e. acetaldehyde (δ 2.20) and
the corresponding gem-diol (δ 1.29), acetic acid (δ 1.88) and
ethanol (δ 1.14).
When we used higher coenzyme concentrations, we were also
able to monitor the changes of concentration for the coenzyme
NAD(P)ϩ (NADϩ C-2 and C-5 protons at δ 9.2 and 9.3 or
NADPϩ C-2 and C-5 protons at δ 9.07 and 9.25, respectively)
and its reduced form NAD(P)H (C-4 and C-3 protons of
NADH at δ 2.6–2.7 and 6.9, respectively and C-4 and C-3
protons of NADPH at δ 2.7–2.8 and 6.9, respectively).
† 1H NMR spectra and graphs for the dismutation of aldehydes with
alcohol dehydrogenases are available as supplementary data. For direct
electronic access see http://www.rsc.org/suppdata/p1/b0/b001039l
We verified the stability of the aldehydes, before any enzymic
assay, by incubating them under the enzyme reaction conditions
DOI: 10.1039/b001039l
J. Chem. Soc., Perkin Trans. 1, 2000, 2283–2287
This journal is © The Royal Society of Chemistry 2000
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Table 1 Acetic acid/ethanol quotient during the dismutation of acet-
aldehyde catalyzed by Saccharomyces cerevisiae ADH (YADH),
Thermoanaerobium brockii ADH (TBADH) or Moraxella sp. TAE123
ADH (MADH)
Conversiona
(%)
[Acetic acid]/
[ethanol]
ADH
pH
YADH
8.8
7.4
8.8
7.4
8.8
7.4
6.0
90
75
78.5
40
63
70
55
1.08
1.1
1.1
1.23
1.6
2.1
TBADH
MADH
1.9
a In the case of acetaldehyde the conversion was calculated from the
sum of the methyl hydrogen absorptions of acetic acid and ethanol,
versus the sum of the methyl hydrogen absorptions of reagents and
products, i.e. acetaldehyde, gem-diol, acetic acid and ethanol.
mesophilic enzyme catalyzes the dismutation of acetaldehyde in
a pH range from 7.4 to 9.0. The fact that YADH displays dis-
mutase activity is in complete agreement with previous reports
that indicated the in vivo participation of yeast ADH in the
metabolism of the ethanol-derived acetaldehyde.17
The thermophilic Thermoanaerobium brockii ADH and the
psychrophilic Moraxella sp. TAE123 ADH were also found to
act as aldehyde dismutases when they reacted with acetalde-
hyde. This is the first example, as far as we know, of ADHs,
derived from extremophilic bacteria, displaying aldehyde dis-
mutase activity. As shown in Table 1, TBADH follows the same
pattern with YADH, producing thus equimolar quantities of
acetic acid and ethanol during the dismutation of acetaldehyde.
In the reaction catalyzed by Moraxella sp. TAE123 ADH, the
ratio of acetic acid and ethanol favors acetic acid formation
(Table 1). The dismutation of acetaldehyde with the psychro-
philic enzyme was also performed at different pH and temper-
ature conditions, resulting always in larger quantities of acetic
acid (Table 1). The excessive formation of acetic acid is attrib-
uted to a second reaction, namely the oxidation of ethanol. To
confirm this assumption, we studied the relative rates of the
reactions of the three ADHs with ethanol and acetaldehyde by
using the same experimental conditions. Both TBADH and
YADH showed a faster turnover number V for the dismutation
of acetaldehyde. The same pattern in kinetic behavior was
previously reported for the dismutation of butyraldehyde com-
pared to the oxidation of butanol with HLADH.5,9 This fact
was attributed to the coupled ping-pong mechanism, which
bypasses the slow steps of enzyme dissociation. It is construct-
ive to emphasize here that, unlike the other ADHs, the
psychrophilic Moraxella sp. TAE123 ADH catalyzes both reac-
tions, production of acetic acid and ethanol, with comparable
rates.
Furthermore, we have also studied the reaction after the
depletion of substrates. The overincubation of the ADHs men-
tioned above with ethanol and acetic acid was studied using
stoichiometric coenzyme concentrations and resulted in the
complete conversion of ethanol to acetic acid. Therefore, it is
evident that these ADHs can catalyze the sequential oxidation
of ethanol to acetic acid. These results provide a further con-
firmation of the mechanism proposed by Oppenheimer and co-
workers.8,11 According to this mechanism aldehyde dismutation
and aldehyde oxidation are part of the same catalytic process
and can be viewed at different times along the assay-progress
curve.
Fig. 1 Part of the 1H NMR time-dependent spectrum showing the
methyl proton absorptions in the dismutation of acetaldehyde catalyzed
by Saccharomyces cerevisiae ADH.
(sodium phosphate buffer pH 6.0 to pH 9.0) without the pres-
ence of enzyme and/or coenzyme. Both acetaldehyde and
propionaldehyde were shown to be stable at all conditions
assayed for time periods longer that those of the enzymic
reaction. The control experiments confirm beyond doubt that
the observed chemical transformations of various substrates
are due to an enzymic reaction and not to a chemical trans-
formation independent of the enzyme.
All three ADHs were shown to act as aldehyde dismutases.
1
Fig. 1 shows the time-dependent H NMR methyl hydrogen
absorptions recorded for the dismutation of acetaldehyde with
YADH. It is clear that the concentrations of acetaldehyde and
the corresponding gem-diol decrease rapidly with the concomi-
tant production of ethanol and acetic acid. The direct 1H NMR
determination of acetic acid concentration present in the
enzymic reaction could not have been observed previously by
UV spectroscopy.18 These results indicate convincingly the
stoichiometry of the dismutation reaction. By integrating the
1
proper H NMR signals, acetic acid and ethanol are produced
in almost equimolar quantities (1.08/1.00) up to 90% conver-
sion (115 min), while this ratio gradually reaches 1.14/1.00 at
95.5% conversion (320 min). These results are in agreement
with the mechanism proposed by Oppenheimer.8,11 The ability
of YADH to catalyze the dismutation of acetaldehyde was also
tested at different pH-values. We conclude (see Table 1) that the
With the use of higher coenzyme concentrations we were
also able to monitor the NADH production. Although in the
mesophilic and thermophilic ADH the duration of the lag
phase was found to be dependent on both acetaldehyde and
enzyme concentration, in the case of the psychrophilic ADH no
lag phase was observed.
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Fig. 2 Part of the 1H NMR time-dependent spectrum showing the methylene proton absorption in the dismutation of propionaldehyde catalyzed by
the psychrophilic Moraxella sp. TAE123 ADH.
The dismutation of aldehydes with the above ADHs was also
studied using propionaldehyde and benzaldehyde as substrates.
In the case of propionaldehyde, the reaction was followed by 1H
NMR integration of the methylene hydrogen absorptions of all
species present in the reaction mixture; namely propionalde-
hyde (δ 2.52) and the corresponding gem-diol (δ 1.55–1.6), pro-
pionic acid (δ 2.12) and propan-1-ol (δ 1.5). All ADHs studied
here were able to catalyze the dismutation of propionaldehyde
with the same catalytic behavior as that in the dismutation of
tations, this type of reaction may be considered as a general
property of alcohol dehydrogenases.
Experimental
Materials and methods
YADH and TBADH were purchased from Sigma Chemical Co.
The psychrophilic Moraxella sp. TAE123 ADH was purified
according to the literature method.15 All other chemicals,
including the oxidized and reduced form of the coenzyme
NAD(P)H, were purchased from Sigma and Aldrich in the
highest available purity.
1
acetaldehyde. It is clear from the stacked portions of the H
NMR spectra, presented in Fig. 2, that the psychrophilic ADH
produces larger quantities of propionic acid than propan-1-ol.
These results were again attributed to the parallel oxidation of
propan-1-ol, which is produced during the dismutation.
1H NMR spectra were recorded in a 500 MHz Bruker AMX
spectrometer. All spectra were acquired in 10% D2O, using
solvent suppression, with solvent presaturation. J-Values are
given in Hz.
In the case of benzaldehyde the direct detection of products
was not possible by using 1H NMR spectroscopy. The reaction
profile was concluded by the detection of the NADH burst,
which accompanies dismutation reactions. For this purpose
higher enzyme concentrations were used. When benzaldehyde
was incubated under standard dismutation conditions in the
absence of enzyme no net production of NADH was detected.
YADH and Moraxella sp. TAE123 ADH were found to
catalyzethedismutationofbenzaldehyde(whilewiththethermo-
philic TBADH no net NADH production was detected).
Standard assay for the dismutation reaction
The standard assay mixture (0.6 ml) for the dismutation reac-
tion consisted of 50 mM sodium phosphate buffer, 15 mM
aldehyde, 1.5 mM NAD(P)ϩ and 10% D2O. The reaction was
initiated by addition of the corresponding alcohol dehydroge-
1
nase and was followed by H NMR spectroscopy (500 MHz)
using solvent suppression with solvent presaturation. For each
alcohol dehydrogenase the quantities of the reagents present in
the reaction mixture were modified in order to achieve comple-
tion of the reaction in a maximum time period of approxi-
mately 12 h.
Conclusion
Our results on the dismutation of aldehydes catalyzed by three
different ADHs conclusively establish that they display alde-
hyde dismutase activity. Since these ADHs belong to different
living organisms, which exhibit different temperature adap-
In the case of acetaldehyde the course of the reaction was
followed by integrating the methyl proton absorptions of all
J. Chem. Soc., Perkin Trans. 1, 2000, 2283–2287
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organic species present in the reaction mixture. Acetaldehyde:
δ 9.64 (1H, q, J 2.9, CH᎐O), 2.20 (3H, d, J 2.9, CH ); gem-diol:
hyde catalyzed by Moraxella sp. TAE123 ADH was per-
formed under the standard assay conditions for dismutation
reactions, using 0.01 U mlϪ1 of Moraxella sp. TAE123 ADH
at pH 8.8, 7.4 and 6.0. The reactions were performed at 30 ЊC
᎐
H
3
δH 5.21 [1H, q, J 5.2, CH(OH)2], 1.29 (3H, d, J 5.2, CH3);
acetate: δH 1.88 (3H, s, CH3); ethanol: δH 3.62 (2H, q, J 7.1,
CH2), 1.14 (3H, t, J 7.1, CH3). In this reaction the conversion
was calculated from the sum of the methyl hydrogen absorp-
tions of acetate and ethanol, versus the sum of the methyl
hydrogen absorptions of reagents and products, i.e. acetalde-
hyde, gem-diol, acetate and ethanol.
1
and 0 ЊC and were followed by H NMR spectroscopy (500
MHz). For the dismutation of acetaldehyde at pH 8.8 1H
NMR spectra were acquired every 2 min until 50% conver-
sion, and then every 30 min until completion of the reaction
(6–8 h).
In the case of propionaldehyde the reaction was followed by
integrating the methylene proton absorptions of all organic
species present in the reaction mixture. Propionaldehyde:
Dismutation of propionaldehyde. The dismutation of propi-
onaldehyde catalyzed by Moraxella sp. TAE123 ADH was
performed under the standard assay conditions for dismu-
tation reactions, using 0.01 U mlϪ1 of Moraxella sp. TAE123
ADH at pH 8.8. The reactions were performed at 30 ЊC and
0 ЊC and were followed by 1H NMR spectroscopy (500
MHz). For the dismutation of propionaldehyde at pH 8.8
1H NMR spectra were acquired every 4 min until 50%
conversion, and then every hour until completion of the
reaction.
δ 9.64 (1H, t, J 1.0, CH᎐O), 2.52 (2H, q, J 7.3, CH ), 1.01 (3H,
᎐
H
2
t, J 7.3, CH3); gem-diol: δH 5.4 [1H, q, J 5.5, CH(OH)2], 1.55
(2H, m, J 7.4, CH2), 0.87 (3H, t, J 7.5, CH3); propionate:
δH 2.12 (2H, q, J 7.6, CH2), 0.97 (3H, t, J 7.6, CH3); propan-1-
ol: δH 3.52 (2H, t, J 6.6, CH2OH), 1.50 (2H, m, J 7.1 CH2), 0.84
(3H, t, J 7.4, CH3). In this reaction the conversion was calcu-
lated from the sum of the methylene hydrogen absorptions of
propionate and propan-1-ol, versus the sum of the methylene
hydrogen absorptions of reagents and products, i.e. propional-
dehyde, gem-diol, propionate and propan-1-ol.
Dismutation of benzaldehyde. The dismutation of benzalde-
hyde catalyzed by Moraxella sp. TAE123 ADH was performed
under the standard assay conditions for dismutation reactions,
using 0.1 U mlϪ1 of Moraxella sp. TAE123 ADH at pH 8.8 and
stoichiometric NADϩ concentration (15 mM). The reaction
was performed at room temperature, and followed by 1H NMR
spectroscopy. The reaction’s outcome was deduced from the
production of NADH (C-4 proton, dd, δ 2.67). For this reac-
Standard assay for the overincubation experiments during the
dismutation of acetaldehyde
During the overincubation experiments the standard assay for
the dismutation reaction was followed, using stoichiometric
coenzyme concentrations. Thus the assay mixture (0.6 ml)
consisted of 50 mM sodium phosphate buffer, 15 mM acetalde-
hyde, 15 mM NAD(P)ϩ and 10% D2O. The reaction was initi-
ated by addition of the corresponding alcohol dehydrogenase
1
tion, H NMR spectra were acquired every hour for a time
period of 12 h.
1
and was followed by H NMR spectroscopy (500 MHz) using
Dismutation reactions catalyzed by TBADH
solvent suppression with solvent presaturation. The reactions
were followed until the depletion of ethanol.
Dismutation of acetaldehyde. The dismutation of acetalde-
hyde catalyzed by TBADH was performed under the standard
assay conditions for dismutation reactions, using 0.1 U mlϪ1 of
Thermoanaerobium brockii ADH at pH 8.8. The reactions were
Dismutation reactions catalyzed by YADH
Dismutation of acetaldehyde. The dismutation of acetalde-
hyde catalyzed by YADH was performed under the standard
assay conditions for dismutation reactions, using 0.1 U mlϪ1 of
Saccharomyces cerevisiae ADH at pH 8.8, 7.4 and 6.0. The reac-
tions were performed at room temperature, and were followed
by 1H NMR spectroscopy. For the dismutation of acetaldehyde
1
performed at 30 ЊC and were followed by H NMR spectro-
scopy (500 MHz). For the dismutation of acetaldehyde at pH
1
8.8 H NMR spectra were acquired every 4 min for 3 h, and
then every hour until completion of the reaction.
1
Dismutation of propionaldehyde. The dismutation of pro-
pionaldehyde catalyzed by TBADH was performed under the
standard assay conditions for dismutation reactions, using 0.1
U mlϪ1 of Thermoanaerobium brockii ADH at pH 8.8. The reac-
at pH 8.8 H NMR spectra were acquired every 2.5 min for
the first 2 h of the reaction, and then every hour until the
completion of the reaction.
1
tions were performed at 30 ЊC and were followed by H NMR
Dismutation of propionaldehyde. The dismutation of pro-
pionaldehyde catalyzed by YADH was performed under the
standard assay conditions for dismutation reactions, using 0.1
U mlϪ1 of Saccharomyces cerevisiae ADH at pH 8.8, 7.4 and
6.0. The reactions were performed at room temperature, and
spectroscopy (500 MHz). For the dismutation of propionalde-
hyde at pH 8.8 1H NMR spectra were acquired every 16 min for
the first 4 h, and then every hour until completion of the
reaction.
1
were followed by H NMR spectroscopy (500 MHz). For the
1
dismutation of propionaldehyde at pH 8.8 H NMR spectra
Acknowledgements
were acquired every 16 min for the first 4 h of the reaction, and
then every hour until the completion of the reaction.
We thank Professor G. J. Karabatsos for his valuable comments.
Dismutation of benzaldehyde. The dismutation of benzalde-
hyde catalyzed by YADH was performed under the standard
assay conditions for dismutation reactions, using 1.0 U mlϪ1 of
Saccharomyces cerevisiae ADH at pH 8.8 and stoichiometric
NADϩ concentration (15 mM). The reactions were performed
References
1 K. Faber, Biotransformations in Organic Chemistry, Springer-
Verlag, Berlin, 1997, pp. 160–206.
2 (a) J. B. Jones, Tetrahedron, 1986, 42, 3351; (b) S. Servi, Synthesis,
1990, 1; (c) W. Hummel and M.-R. Kula, Eur. J. Biochem., 1989, 184,
1.
3 H. Sund and M. Theorell, Alcohol Dehydrogenase, in The Enzymes,
ed. P. D. Boyer, H. Lardy and K. Myrback, Academic Press,
New York, 2nd edn., 1960, vol. VII, pp. 25–83.
1
at room temperature, and were followed by H NMR spectro-
scopy. The reaction’s outcome was deduced from the produc-
tion of NADH (C-4 proton, dd, δ 2.6). For this reaction,
1H NMR spectra were acquired every hour for a time period of
12 h.
4 R. H. Abeles and H. A. Lee, J. Biol. Chem., 1960, 235, 1499.
5 K. Dalziel and F. M. Dickinson, Nature, 1965, 206, 255.
6 N. K. Gupta and W. G. Robinson, Biochim. Biophys. Acta, 1966,
118, 429.
Dismutation reactions catalyzed by Moraxella sp. TAE123 ADH
Dismutation of acetaldehyde. The dismutation of acetalde-
7 F. M. Dickinson and G. P. Monger, Biochem. J., 1973, 131, 261.
2286
J. Chem. Soc., Perkin Trans. 1, 2000, 2283–2287

View Article Online
8 G. T. M. Henehan and N. J. Oppenheimer, Biochemistry, 1993, 32,
735.
9 G. L. Shearer, K. Kim, K. M. Lee, K. Wang and B. V. Plapp,
Biochemistry, 1993, 32, 11186.
10 L. P. Olson, J. Luo, Ö. Almarsson and T. C. Bruice, Biochemistry,
1996, 35, 9782.
11 G. T. M. Henehan, S. H. Chang and N. J. Oppenheimer,
Biochemistry, 1995, 34, 12294.
15 I. Tsigos, K. Velonia, I. Smonou and V. Bouriotis, Eur. J. Biochem.,
1998, 254, 356.
16 (a) V. Prelog, Pure Appl. Chem., 1964, 9, 119; (b) R. MacLeod,
H. Prosser, L. Fikentscher, J. Lanyi and H. S. Mosher, Biochemistry,
1964, 3, 838.
17 J. Thielen and M. Ciriacy, J. Bacteriol., 1991, 173, 7012.
18 S. Trivic´, V. Leskova and G. W. Winston, Biotechnol. Lett., 1999, 21,
231.
12 S. Svensson, A. Lundsjö, T. Cronholm and J.-O. Höög, FEBS Lett.,
1996, 394, 217.
13 K. Nakamura, T. Miyai, J. Kawai, N. Nakajima and A. Ohno,
Tetrahedron Lett., 1990, 31, 1159.
14 R. Lamed and J. G. Zeikus, Biochem. J., 1981, 195, 183.
19 R. Lamed and J. G. Zeikus, J. Bacteriol., 1980, 141, 1251.
20 M. Peretz, O. Bogin, E. Keinan and Y. Burstein, Int. J. Pept. Protein
Res., 1993, 42, 490.
21 K. Velonia, I. Tsigos, V. Bouriotis and I. Smonou, Bioorg. Med.
Chem. Lett., 1999, 9, 65.
J. Chem. Soc., Perkin Trans. 1, 2000, 2283–2287
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