FEMS Microbiology Letters 125 (1995) 83-88
Occurrence of the methylglyoxal bypass in halophilic Archaea
Aharon Oren
*, Peter
Gurevich
Division of Microbial and Molecular Ecology, The Alexander Silverman Institute of Life Sciences, and The Moshe Shilo Center for Marine
Biogeochemistry, The Hebrew Universiry of Jerusalem, Jerusalem 91904, Israel
Received 28 September 1994; accepted 13 October 1994
Abstract
Eight species of halophilic Archaea were tested for the presence of the enzymes of the methylglyoxal
bypass.
Methylglyoxal synthase was found in extracts of all species tested, with the exception of Halobacterium salinarium and
Halobacterium cutirubrum. The enzyme of Haloferax volcanii was most active at pH 7 in the absence of salt, and in the
presence of 3 M NaCl or KC1 activity was half of that without salt, and was inhibited by phosphate. Glyoxalase I was
detected in all species tested. Optimal activity of H. volcanii glyoxalase I was found at pH 7 and 3 M KCl; in the absence of
salt, activity was strongly reduced. Glutathione could be replaced by y-glutamylcysteine
as the acceptor of the D-lactoyl
group. The work shows that the methylglyoxal bypass may be operative in representatives of the archaeal kingdom.
Keywora’s: Methylglyoxal
synthase; Glyoxalase
I; Halophilic
1. Introduction
When grown in the presence of sugars or glycerol, many halophilic Archaea, notably those belonging to the genera Haloferax and Haloarcula, form
different acids. Dependent on the species tested,
mixtures of D-lactate and acetate or pyruvate and
acetate were found. Formation of the same acids
could also be demonstrated upon addition of micromolar concentrations of glycerol to brines inhabited
by halophilic archaeal communities [l].
D-Lactate can be formed by halophilic Archaea in
different ways. Two types of D-lactic dehydrogenase
were identified in the past in this group of organisms: an NAD+-dependent enzyme 121, and an enzyme using ferricyanide as electron acceptor [3].
* Corresponding author.
0378-1097/95/$09.50
archaea; y-Glutamylcysteine;
Another way D-lactate may be formed by bacteria
is through the methylglyoxal bypass [4,5]. In this
pathway, methylglyoxal is produced from dihydroxyacetone phosphate by methylglyoxal synthase (EC
4.2.99.11). Methylglyoxal is then converted to Dlactate by the operation of glyoxalase I (Slactoylglutathione methylglyoxal-lyase; EC 4.4.1.51,
which forms S-iactoylglutathione from methylglyoxal and reduced glutathione, followed by the action
of glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.61, which hydrolyses S-lactoylglutathione to glutathione and D-lactate.
The function of the methylglyoxal bypass in cell
metabolism is still not completely understood. One
of the functions is probably to enable the formation
of acetyl-CoA from dihydroxyacetone phosphate under conditions where low phosphate concentrations
limit the activity of glyceraldehde 3-phosphate dehydrogenase. In accordance with this role, methylgly-
Q 1995 Federation of European Microbiological Societies. All rights reserved
SSDI 0378-1097(94100477-3
Archaea
A. Oren, P. Gurevich / FEMS Microbiology Letters 125 (1995) 83-88
84
oxal synthase is inhibited by inorganic phosphate [4].
The enzymes of the methylglyoxal bypass have been
detected in eukaryotes such as yeast, and in several
prokaryotes, notably Escherichia coli [5] and other
members of the Enterobacteriaceae,
and several
members of the genera Pseudomonas
[4,6] and
Clostridium [7,8]. It has been claimed that the methylglyoxal bypass is a metabolic pathway fundamental to life [9]. However, no reports exist on the
occurrence of the enzymes of the methylglyoxal
bypass in the archaeal kingdom, in spite of the many
studies on the variety of metabolic pathways operating within this group [lo-121.
In order to test whether the activity of the methylglyoxal bypass may also lead to the formation of
D-lactate in halophilic Archaea, we examined the
presence and properties of methylglyoxal
synthase
and glyoxalase I in eight species of the Halobacteriaceae, belonging to different genera.
2. Materials and methods
2.1. Bacterial strains and culture conditions
The following strains were used in this study:
Halobacterium cutirubrum NRC 34001, Halobacterium salinarium strain 5, Halobacterium saccharovorum ATCC 29252, Haloferax volcanii ATCC
29605,
Haloferax
mediterranei
ATCC
35300,
Haloferax denitrificans ATCC 35960, Haloarcula
marismortui ATCC 43049, and Haloarcula vallismortis ATCC 29715.
Table 1
Activities
of methylglyoxal
synthase
Strain
Halobacterium salinarium
Halobacterium cutirubrum
Halobacterium saccharovorum
Haloferar volcanii
Haloferar mediterranei
Haloferax denitrijicans
Haloarcula marismortui
Haloarcula vallismortis
and glyoxalase
Cultures (500 ml portions in l-l Erlenmeyer flasks)
were grown in a rotatory shaker at 35°C in the
following media (g l- ’ ): for H. cutirubrum and H.
salinarium: NaCl, 250; KCl, 5; MgCl, * 6H,O, 5;
NH,Cl, 5, and yeast extract, 10. The medium for H.
volcanii, H. mediterranei, H. denitrijkans, and H.
saccharovorum
consisted of: NaCl, 175; MgCl, .
6H,O, 20; K,SO,, 5; CaCl,. 2H,O, 0.1, and yeast
extract, 5. The medium for H. marismortui and H.
vallismortis contained: NaCl, 206; MgSO, . 7H,O,
36; KCl, 0.37; CaCl, . 2H,O, 0.5; MnCl,, 0.013,
and yeast extract, 5. All media were adjusted to pH
7.0 with NaOH. In part of the experiments, media
were supplemented
with 1 g glycerol 1-r; yeast
extract concentrations were then lowered to 1 g ll’,
and PIPES buffer was added from a separately sterilized solution to a final concentration of 20 mM.
2.2. Enzymatic assays
Cells at the late exponential growth phase were
collected by centrifugation, and disrupted by sonication in the cold in buffer (3 M KC1 + 100 mM
PIPES, pH 7, for assay of methylglyoxal synthase, 3
M KC1 + 100 mM K-P04, pH 6.8, for assay of
glyoxalase I). Cell debris was removed by centrifugation (10 min, 12000 X g>, and the supematant
fraction, containing typically between 10 and 30 mg
was used in the enzymatic assays.
protein ml-l,
Protein was determined by the Lowry method, using
bovine serum albumin as standard.
The reaction mixture for the assay of methylglyoxal synthase contained 0.05 or 0.1 ml cell extract,
I in cell extracts of halophilic
Archaea
Methylglyoxai synthase activity
(nmol (mg protein)-’
min- ‘1
Glyoxalase I activity
(nmol (mg protein) -
0 (5)
0 (3)
11.0 f 0.7 (2)
30.0 + 6.5 (7)
9.3
7.1
13.2
11.5
17.0 + 5.3 (5)
19.3
4.7 f 2.8 (4)
16.0 + 3.4 (3)
11.7 + 1.4 (2)
19.9
9.8 + 1.0 (2)
7.5 * 1.5 (2)
’ min- ’ 1
Activities were measured at 35°C in the presence of 3 M KC1 + 100 mM PIPES, pH 7 (methylglyoxal synthase) or 3 M KC1 + 100 mM
K-PO, buffer pH 6.8 (gfyoxalase I). Mean values and standard deviations are given, numbers in parentheses stating the number of
independent experiments. When no standard deviation was given, values were based on a single experiment.
85
A. Oren, P. Gureuich / FEMS Microbiology Letters 125 (1995) 83-88
0.85 ml buffer containing 3 M KC1 and 100 mM
PIPES, adjusted to pH 7.0 with NaOH, and 0.05 ml
of 40 mM dihydroxyacetone phosphate (lithium salt),
in a final volume of 1 ml. The reaction mixture for
the assay of glyoxalase I contained 0.05 or 0.1 ml
cell extract, 0.85 ml buffer containing 3 M KCl, 100
mM KI-I,PO, and 2 mM glutathione, adjusted to pH
6.8 with KOH, and 0.05 ml of 10 mM methylglyoxal, in a final volume of 1 ml. After different times
of incubation at 35°C 0.1~ml samples were withdrawn, and assayed for methylglyoxal. The reaction
mixtures were modifed as indicated with respect to
salt (NaCl or KCl) content of the buffers, pH, and
addition of other compounds.
Methylglyoxal was assayed calorimetrically [5].
Samples (0.1-0.2 ml) were diluted with water to a
final volume of 1 ml. After addition of 0.33 ml of
0.1% 2,4-dinitrophenylhydrazine in 2 N HCl, followed by 10 min incubation at 35°C 1.67 ml 10%
NaOH was added. After 15 min incubation at room
temperature the OD was measured at 550 nm.
3. Results and discussion
Methylglyoxal synthase activity was detected in
extracts of six out of the eight species of Halobacteriaceae tested. Activities were proportional to the
amount of cell extract added. Activities between 7.1
and 30 nmol mg protein-’ min-’ were measured at
35°C (Table 1). No methylglyoxal synthase activity
could be shown in Halobacterium species (H. salinarium and H. cutirubrum). The enzyme was present in all species of the genera HaZoferax and
Haloarcula examined, and also in Halobacterium
saccharovorum, a species which, though presently
classified in the genus Halobacterium, differs greatly
from the type species of the genus, and awaits a
taxonomic reappraisal 1131. It is unknown whether
the lack of detectable methylglyoxal synthase activity in H. salinarium and H. cutinrbrum is due to the
fact that these organisms do not possess the enzyme,
or whether the assay conditions used were not suitable to detect it.
The properties of methylglyoxal synthase of
Haloferax volcanii were investigated in more detail.
Highest activity was found at pH 7 in the absence of
salt. In the presence of 3 M NaCl or KC1 activity
6ol
0
SALT
CONCENTNATION60
56789
PH
Fig. 1. Effect of salt concentration (left panel) and pH (right
panel) on the activity of methylglyoxal synthase of Huloferax
wok&i. The effect of NaCl(0) and KCl(0) concentration was
measured at pH 7.0 (using 100 mM PIPES buffer). Reaction
mixtures with NaCl contained 0.3 M KCl in addition. The effect
of pH was measured in reaction mixtures without added KCl, and
containing 50 mM buffer (Tris for pH 8 and 9, PIPES for pH 7
and 6, and MES for pH 5).
was half that without salt added to the reaction
mixture (Fig. 1). The inhibitory effect of high salt
concentrations on the enzyme from H. volcanii is
unusual: most enzymes characterized in members of
the Halobacteriaceae are optimally active in the presence of high salt concentrations, and in the absence
of salt they lose their activity and stability [3]. Phosphate proved to be a potent inhibitor, and a concentration of 1 mM reduced activity by half, and hardly
any activity was found above 5 mM. Inorganic pyrophosphate, reported to have a similar inhibitory
action as phosphate [4], was only slightly inhibitory
(40% inhibition at 8 mM). The activity of methylglyoxal synthase in H. volcanii was not enhanced when
cells were grown in the presence of glycerol. Likewise, extracts of H. salinarium grown in medium
supplemented with 1 g glycerol 1-l did not show
methylglyoxal synthase activity.
Glyoxalase I activity was detected in all Halobacteriaceae tested. Activities were proportional to the
amount of cell extract added, and values between 4.7
and 19.9 mnol mg protein-’ min-’ were measured
at 35°C (Table 1). Activities in H. salinarium and
H. volcanii were similar in extracts of cells grown in
the absence and in the presence of 1 g glycerol 1-l.
Also in E. coli the glyoxalase I activity (12 nmol mg
protein-l min-‘, in the same order of magnitude as
A. Oren, P. Gurecich / FEMS Microbiology Letters 125 (1995) 83-88
86
10
0
5
SALT CONCENTRATION
(Mb
6
7
a 9
PH
Fig. 2. Effect of salt concentration
(left panel) and pH (right
panel) on the activity of glyoxalase I of Huloferar volcanii. The
effect of NaCl (0) and KC1 (0) concentration was measured at
pH 6.8 (using 0.1 M phosphate buffer). Reaction mixtures with
NaCl contained in addition 0.15 M KCl. The effect of pH was
measured in reaction mixtures with 3 M KCl, and containing 50
mM buffer (Tris for pH 8 and above, phosphate for pH 7 and
below).
the activities reported in the present study) was not
enhanced when glycerol was present in the growth
medium. However, in the yeast Succharomyces cereuisiue the activity of glyoxalase I was highly increased when grown in the presence of glycerol
[9,14]. Optimal activity of Huloferux uolcunii glyoxalase I was found at pH 7 in the presence of 3 M
KCI; NaCl supported activity to a lesser extent. In
the absence of salt activity was strongly reduced
(Fig. 2).
In the absence of added glutathione no activity
was detected. Glutathione could be replaced by yglutamylcysteine,
which was reported to be the major low-molecular mass thiol in halobacteria, rather
than glutathione [15]. The rate of disappearance of
methylglyoxal by H. uolcunii extracts was higher in
the presence of y-glutamylcysteine
than in the presence of glutathione. However, at least to some extent
the effect was due to the occurrence of a non-enzymatic reaction.
It has been suggested that the enzymes of the
methylglyoxal bypass may be universally present in
all living organisms [9]. However, the variety of
types of prokaryotes tested for the presence of the
pathway is still rather limited; to our knowledge
activity of methylglyoxal
synthase and glyoxalase I
has only been demonstrated in a few representatives
of the proteobactera [4-6,161 and clostridia [7,8].
The present work shows that the first two enzymes
of the pathway are present in the halophile branch of
the archaeal kingdom as well. No attempts were
made in this study to assay for glyoxalase II.
The results presented here add an additional pathway of carbon flow between glucose and acetyl-CoA
in halophilic Archaea. The diversity in this part of
cell metabolism now includes the activity of different portions of the Embden-Meyerhof
pathway
[12,17,18], in addition to a modification of the Entner-Doudoroff pathway of carbohydrate degradation
[10,11,17], and the potential activity of the methylglyoxal bypass between dihydroxyacetone
phosphate
and D-lactate. To what extent the enzymatic activities documented here contribute to the formation of
D-lactate in cultures and natural communities
of
halophilic Archaea cannot as yet be ascertained. The
activity of the methylglyoxal
bypass is probably
regulated by the intracellular phosphate concentration, as phosphate strongly inhibits methylglyoxal
synthase. At least one study shows that natural communities of halobacteria in the Dead Sea may be
phosphate-limited
[19], suggesting that the methylglyoxal bypass may be of importance to communities of halophilic Archaea in their natural habitat.
Acknowledgements
We thank Gerhard Gottschalk (GBttingen) for invaluable discussions. This work was supported by
grants from the Ministry of Science and Culture,
Land Niedersachsen, and the Israel Science Foundation administred by the Israel Academy of Sciences
and Humanities.
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