From the Laboratoire de Biotechnologie, FRE-CNRS 2230 Biocatalyse, Faculté des Sciences et des Techniques,
Université de Nantes, 44322 Nantes Cedex 3 France, the
§ Jean-Marie Wiame Institute for Microbiological Research,
1, Av. E. Gryson, B-1070 Brussels, Belgium, and the ¶ Laboratory
for Genetics and Microbiology, Vrije Universiteit Brussel and
Department of Microbiology, Flanders Interuniversity Institute for
Biotechnology, 1, Av. E. Gryson, B-1070 Brussels, Belgium
Received for publication, January 16, 2001, and in revised form, March 15, 2001
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ABSTRACT |
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In Bacillus stearothermophilus
ornithine acetyltransferase is a bifunctional enzyme, catalyzing
the first and the fifth steps of arginine biosynthesis; it follows a
ping-pong kinetic mechanism. A single chain precursor protein is
cleaved between the alanine and threonine residues in a highly
conserved ATML sequence leading to the formation of Ornithine acetyltransferase
(OATase,1
N2-acetyl-L-ornithine:
L-glutamate N-acetyltransferase; EC 2.3.1.35,
encoded by the argJ gene in procaryotes) participates in
arginine biosynthesis in microorganisms (for a review see Ref. 1). The
OATase-mediated transacetylation follows a ping-pong kinetic mechanism
in which the enzyme is acetylated as an intermediate protein from which the acetyl group is subsequently transferred to glutamate (2). In the
thermophilic bacteria Bacillus stearothermophilus and
Thermotoga neapolitana, in the thermophilic archaeon
Methanococcus jannaschii (2), and in Saccharomyces
cerevisiae (3), OATase has been shown to be synthesized as a
precursor protein that undergoes proteolytic cleavage, leading to the
formation of Posttranslational autoproteolysis has been described for several
proteins from eucaryotes and procaryotes (6). In glycosylasparaginase from Flavobacterium, structural and biochemical studies have
shown that cleavage results from a nucleophilic threonine attack
on the peptide linkage with the preceding aspartate residue (7, 8). In
addition to threonine the deprotonated side chains of cysteine and
serine residues, are also able to attack a preceding carbonyl group
resulting in N Thus the question arises as to how the cleavage of the OATase precursor
protein is accomplished in procaryotes. To answer this question
we have investigated the thermostable OATase from B. stearothermophilus. Our data indicate that precursor protein cleavage is necessary for enzyme activation and that the invariant threonine Thr-197 is responsible for the self-catalyzed cleavage reaction.
Strains and Materials--
The argJ gene was cloned
from the B. stearothermophilus NCIB 8224 strain (11). OATase
from B. stearothermophilus is able to accept acetyl donors
both from acetyl-CoA and
N-acetyl-L-ornithine and therefore catalyzes the
first (argA) and fifth (argJ) steps in arginine
biosynthesis (12). Escherichia coli K12 strains XA4
(F
Oligonucleotides for site-directed mutagenesis were purchased from Life
Technologies, Inc. and the nickel-NTA affinity resin was
purchased from Qiagen. D. Gigot (Brussels, Belgium) kindly provided
14C-labeled N-acetyl-L-glutamic acid.
Site-directed Mutagenesis and Recombinant DNA
Constructions--
Mutations were introduced in the B. stearothermophilus argJ gene by polymerase chain reaction as
previously described for the argR gene (15). In pargJ-Bs/N
plasmid the argJ gene was fused to an N-terminal His tag
encoding sequence (2), and the ACG codon (threonine at a 197-position)
was mutated to either AGT (serine), TGT (cysteine) or GGT (glycine).
Sequences of mutagenic oligonucleotide primers are available on
request. The mutated argJ sequences were cloned in pCR4.1
TOPO vectors (Invitrogen). The pargJ-Bs/N and the pargJ-Bs/C plasmid
(2) were also used to construct shortened argJ coding only
for the
We also constructed an argJ variant with a translation stop
codon for the Molecular Mass Determination of Mutant OATases--
Recombinant
E. coli XS1D2 cells carrying corresponding plasmids were
cultivated in arginine-less minimal medium, and His-tagged proteins
were then purified using Ni2+-affinity column. Molecular
masses of purified proteins were estimated by gel permeation
chromatography using a TSK-3000SW column (Merck) directly on-line with
the HPLC system (Merck) using Tris buffer 0.01 M (pH 7.5 with NaCl 0.15 M) as the mobile phase at a flow rate of 1 ml/min. Detection of proteins was carried out at 280 nm, and molecular
masses were calculated using standard protein markers (Amersham
Pharmacia Biotech): aldolase (158 kDa), bovine serum albumin (67 kDa),
chicken ovalbumin (44 kDa), chymotrypsinogen (25 kDa), and ribonuclease
(13.7 kDa). SDS-PAGE of proteins was carried out as described by
Ausubel et al. (17). Gels were stained with Coomassie
Brilliant Blue R-250 (Bio-Rad). Protein concentration was determined by
the Bradford method (18) using bovine serum albumin as standard.
Enzymatic Assays--
A previously developed HPLC-based method
was used to evaluate quantitatively OATase-catalyzed formation of
N-acetyl-L-glutamate (2). Enzyme activity was
measured in a mixed buffer (MES 0.1 M, PIPES 0.1 M, Tris 0.1 M, glycine 0.1 M, and
K2HPO4 0.1 M, at pH 7.0) containing
L-glutamate (20 mM) and
N2-acetyl-L-ornithine (20 mM). After 2 min of preincubation at 60 °C the reaction
was initiated by adding the purified enzyme. The reactions were
continued from 5 min to 24 h at 60 °C and then stopped by
adding 500 mM phosphoric acid. One enzyme unit is defined as the amount of enzyme producing 1 µmol of
N-acetyl-L-glutamate/min.
Detection of Acetylated Proteins--
Purified His-tagged
OATases were incubated with 14C-labeled
N-acetyl-L-glutamic acid as acetyl donor, but in
the absence of acetyl acceptor (semi-reaction) in the above mentioned
mixed buffer at 37 °C for 30 min or at 65 °C for 1 h,
reaction products were then separated on SDS-PAGE (a 12% gel). After
coloration with Coomassie Brilliant Blue, gels were treated with an
amplifier solution (Amersham Pharmacia Biotech), and radioactive bands
were visualized by autoradiography using Hyperfilm TM (Amersham
Pharmacia Biotech).
Measurement of CoA Formation--
In a previous study we noticed
that a rate of acetylated intermediates both for B. stearothermophilus and T. neapolitana OATases was lower
as compared with the consumption of [14C]acetyl-CoA or
[14C]acetyl-L-glutamate substrates in a
semi-reaction (2). To find out the matter we performed semi-reaction by
quantification of the deacetylated donor. The reaction was carried out
in a 200-µl incubation mixture containing the above-mentioned mixed
buffer, 0.5 µmol of acetyl CoA, and 5 µmol of glutamate. The
reaction was initiated by the addition of a purified His-tagged OATase. After 20 min of incubation at 60 °C the reaction was stopped by cooling on ice, and the samples were immediately filtered on Microcon 10 (Amicon) to remove the enzyme. Quantitative determination of CoA was
carried out on 100 µl of filtrate by reverse phase HPLC analysis
using an Apex ODS 3 µ column (250 × 4.6 mm, from Jones Chromatography, Inc.) as described in (19).
In Vitro Synthesis of OATase--
In vitro synthesis
of wild-type or mutant proteins was carried out in S30 extracts of
E. coli BL21 prepared as described (20) with minor
modifications using circular or linear DNA templates. T7 RNA polymerase
was purchased from Promega and
L-[35S]methionine was from Amersham Pharmacia
Biotech. Where indicated a mixture of protease inhibitors (Sigma)
against metalloproteases, serine-, cysteine-, and aspartate-proteases,
was added to the reaction mixture. The samples were treated at 65 °C
for 10 min and then shortly centrifuged, and the supernatant was used
for protein separation on SDS-PAGE.
Separately Expressed Subunits of OATase Are Not
Active--
We previously showed that the two subunits of
recombinant OATases from three thermophilic microorganisms, always
co-purified by affinity chromatography irrespective of the N- or
C-terminal His tag, respectively, fused to the
We amplified and cloned separately B. stearothermophilus DNA
argJ regions corresponding to
These data were confirmed by studying plasmid constructions in which
the two subunits were independently translated from a common mRNA
(see Fig. 1B). Indeed, no complementation was observed in
E. coli XS1D2 and XA4 mutants, and no OATase activity was
detected in cell extracts of the transformants. Thus, the data
show that independently synthesized Analysis of Thr-197-substituted Mutant Proteins in
Vitro--
Assuming that the invariant threonine (Thr-197 in B. stearothermophilus OATase) in the conserved ATML sequence could
play an essential role in the intramolecular cleavage we replaced this amino acid by a serine, cysteine, or glycine residue. The plasmids carrying these replacements were used as DNA templates to study the
cleavage capacity of the precursor proteins in E. coli S30 extracts.
On SDS-PAGE a wild-type B. stearothermophilus OATase was
detected as two bands corresponding to the
In contrast to the wild-type argJ gene, the three T197S,
T197C, and T197G mutants produced a low migrating band corresponding to
the precursor protein (see Fig. 2A). However, in addition, two bands corresponding to the Kinetics of Cleavage of Mutant OATase Precursor
Proteins--
SDS-PAGE analysis of purified His-tagged wild-type and
threonine-substituted mutant proteins synthesized in E. coli
cells confirmed the results of the in vitro experiments
except that the 37-kDa protein was not detected (Fig. 2B).
The molecular mass of the native wild-type OATase was determined as
90-100 kDa by gel permeation as expected for a heterotetrameric
structure (2). However, molecular mass determination of purified T197C
and T197S mutant proteins gave major protein peaks of 54 kDa and
90-100 kDa corresponding to the precursor and the heterotetrameric
molecules, respectively. Only the precursor form of 54 kDa was detected
with the T197G mutant.
The mutant proteins were diluted in Tris buffer 10 mM, pH
8.0, to a concentration of 1 mg/ml and incubated at 50 °C for
24 h, taking samples regularly to visualize the kinetics of
subunits formation by SDS-PAGE (Fig. 3).
The T197S mutant precursor underwent the transition to OATase Activity of Thr-197-substituted Mutant
Proteins--
Purified His-tagged proteins were tested for OATase
activity at 50 °C. A specific activity of 29 µmol of
N-acetyl-L-glutamate/min/mg protein was measured
for the wild-type enzyme, whereas no activity was detected for the
T197G mutant. A very low activity, 0.1 µmol of
N-acetyl-L-glutamate/h/mg protein was detected
for freshly purified T197S and T197C mutant proteins. Moreover, T197S
and T197C mutant proteins exhibited a similar low OATase activity after incubation at 10 °C for a week, although under these
conditions the mutant precursor proteins underwent complete cleavage as
judged by SDS-PAGE.
In agreement with these data none of the three threonine-substituted
argJ mutants supported the growth of E. coli
XS1D2 (argE) and XA4 (argA) strains in a medium
devoid of arginine. Thus the low level of OATase activity exhibited by
T197S or T197C mutants is not sufficient to complement E. coli
argE or argA mutant strains.
Acetyl Enzyme Intermediate Formation--
We performed a
semi-reaction in the presence of only 14C-labeled
N-acetyl-L-glutamic acid to determine which
subunit is acetylated in OATase. The single band corresponding to the
We quantified also the B. stearothermophilus OATase-mediated
formation of CoA from non-labeled acetyl-CoA in the absence and presence of L-glutamate as an acetyl group acceptor (Table
I). About 5 mol of coenzyme A were
produced per mole of enzyme in the absence of glutamate suggesting that
hydrolysis of the acetyl enzyme intermediate to produce acetate
under the conditions used.
Our previous studies on three procaryotic OATases indicated
autoproteolysis as a likely mechanism for the intramolecular cleavage of the OATase preprotein into Given the crucial role of the invariant threonine in intramolecular
cleavage of preOATase, we assume that the processing of a wild-type
OATase precursor from B. stearothermophilus is similar to
the processing of glycosylasparaginase from Flavobacterium (8, 22). When the de novo synthesized OATase precursor is correctly folded, the hydroxyl group of Thr-197 can be deprotonated and
its nucleophilicity become enhanced (Fig.
6). A nucleophilic attack on the
and
subunits that assemble into a heterotetrameric 2
2
molecule. The
subunit has been shown to form an acetylated intermediate in the
course of the transacetylation reaction. The present data show that the
precursor protein synthesized in vitro or in
vivo undergoes a self-catalyzed cleavage involving an invariant threonine (Thr-197). Using site-directed mutagenesis T197G,
T197S, and T197C derivatives have been generated. The T197G
substitution abolishes both precursor protein cleavage and catalytic
activity, whereas T197S and T197C substitutions reduce precursor
cleavage and catalytic activity in the order Thr-197 (wild type)
Ser-197
Cys-197. A mechanism is proposed in which Thr-197 plays a
crucial role in the autoproteolytic cleavage of ornithine acetyltransferase.
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ABSTRACT
INTRODUCTION
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DISCUSSION
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and
subunits that assemble into heterodimeric or
heterotetrameric molecules in eucaryotes or procaryotes, respectively.
The N-terminal amino acid residue of the
subunit of mature OATase
has been shown to be a threonine and cleavage shown to occur between
the alanine and threonine residues in a conserved ATML sequence of the
precursor proteins (2-4). Cleavage of the yeast OATase precursor has
been suggested to be self-catalyzed rather than protease-assisted
(5).
O or N
S shift (8, 9). The peptide amide bond
is therefore replaced with a more reactive (thio)ester bond, and a
subsequent attack by a second nucleophile, which can be an activated
water molecule, breaks the ester bond thereby exposing Thr, Cys, or Ser
at the N terminus of the downstream peptide (10).
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argA nalA
s hsdR) and XS1D2R (F
(ppc-argE) nalA rpoB
hsdR recA)) were used in
complementation tests to check the functionality of B. stearothermophilus argJ derivatives in E. coli cells as described previously (13). The E. coli BLR(DE3) was used to overexpress the B. stearothermophilus mutant argJ
gene. The separate expression of
and
subunits of OATase was
carried out from the strong PargC promoter of B. stearothermophilus (14) in E. coli K802
(F
metB).
or
subunit fused to terminal His tags (Fig. 1). To
evaluate the role of Thr-197 as an N-terminal residue, two versions of
the
subunit were designed: (i) an N-terminal methionine was added
upstream of Thr-197 (argJ-
1), or this Thr-197 was
replaced by a methionine (argJ-
2; see Fig. 1A). argJ sequences encoding
or
subunit
were fused to the PargC promoter by the "overlapping
extension" method (16) and cloned in the EcoRI site of pBR322.
subunit encoding sequence immediately followed by a
ribosome-binding site preceding either
1 or
2 versions (see
above). This preserved the single mRNA transcription but resulted
in independent translation of the
and
subunits (see Fig.
1B). All mutations were confirmed by DNA sequencing.
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or the
subunit
(2). The question arose whether
and
subunits, when synthesized independently, could exhibit enzymatic activity when mixed together in vitro or coexpressed in vivo.
and
subunits (two
versions,
1 and
2, were obtained for
, differing by their
N-terminal sequences, MTMLA and MMLA, respectively; see Fig.
1A). None of the plasmids
carrying the shortened argJ gene, namely
argJ-
, argJ-
1, or argJ-
2, was
able to complement the E. coli XS1D2 and XA4 strains
deficient for argE and argA genes, respectively. Additionally, none of the purified truncated proteins used either separately or mixed in equimolar concentrations as
and
1 or
and
2 subunits exhibited OATase activity after 24 h of
incubation at 37 °C or 50 °C. Moreover, when separately expressed
and then mixed together,
and
1 or
and
2 subunits never
assembled as judged from PAGE performed in non-denaturant conditions
(data not shown).
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Fig. 1.
Designing plasmid constructions to produce
separately and
subunits of B. stearothermophilus OATase.
A, two subunit coding regions were cloned separately in two
plasmids; amino acids located in an N-terminal region of
1 or
2
subunit versions are indicated. B, two subunit coding
regions are transcribed as a common mRNA but expressed as
independent proteins. The nucleotide and amino acid sequences for a
wild-type and two
-
1 and
-
2 constructions are shown. The
created sequences between
and
1 or
and
2 coding regions
are shown in italic; the Shine-Dalgarno site is
underlined.
and
subunits (irrespective
of the N-terminal regions created for the latter) do not reconstitute a
functional B. stearothermophilus OATase in E. coli host cells.
and
subunits,
indicating that intramolecular cleavage is a very rapid and efficient
process in vitro (Fig.
2A). Added protease inhibitors
did not increase the yield of subunit formation; on the contrary, the
intensity of the subunit bands decreased and smaller protein appeared.
The protease inhibitors mixture was therefore omitted in further
experiments.
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Fig. 2.
Synthesis of the wild-type B. stearothermophilus OATase and threonine-substituted mutant
proteins in vitro
(A) and in E. coli
cells (B). A, lanes
1 and 3, a wild-type OATase in absence and lane 2, in
presence of a protease inhibitors mixture; lanes 4, 5 and
6, mutant proteins T197G, T197S, and T197C, respectively.
B, lane M, molecular mass markers; wt,
the purified His-tagged wild-type OATase; T197G, T197C, and T197S, the
purified His-tagged mutant proteins without further incubation (0 h)
and after 21 h of incubation in Tris buffer 10 mM, pH
8.0, at 50 °C.
and
subunits were still present in protein samples synthesized from the T197S and T197C mutant DNA
templates; this was not the case for the T197G mutant. In these
experiments we noticed that when mutant genes were used as DNA
templates, a weak 37-kDa protein band that could be attributed to
translation from a potential internal ribosome-binding site in mutant
argJ mRNA was present as well.
and
subunits faster than did the T197C mutant. Densitometric analysis of
the precursor protein and subunit protein bands formed over a 24-h
incubation showed that the kinetics of hydrolysis followed a first
order curve and that the apparent half-life of the precursor was 71 min
(k1 = 1.1 10
2
min
1) and 35 h (k2 = 2.0 10
2 h
1) for the
T197S and the T197C mutant proteins, respectively. The subunit bands
did not appear with the T197G mutant confirming its inability to
undergo intramolecular cleavage (data not shown). We also found
that the ArgJ-T197S mutant precursor was processed throughout the 3.5 to 9.5 pH range and with a maximal rate at 69 °C (Fig.
4).
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Fig. 3.
Kinetics of intramolecular cleavage of
purified mutant precursor proteins at 50 °C. The used buffer
was 10 mM Tris, pH 8.0. A, lane 1,
molecular mass markers; lanes 2, 3, 4, 5, 6, 7, 8 and 9, the T197S mutant protein sample after 0, 10, 20, 30, 60, 90, 120, and 180 min of incubation; lane 10, after
21 h of incubation. B, lanes 1, 2, 3, 4, 5, 6, 7 and 8, the T197C mutant protein sample after
0, 3, 6, 9, 12, 15, 18, and 21 h of incubation. C
and D, densitometric evaluation of a precursor
protein band disappearance as a function of the incubation time for
T197S and T197C, respectively. The relative efficiency of the cleavage
is shown as a log% of the precursor protein band synthesized de
novo. Analysis was carried out by a 12% SDS-PAGE.
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Fig. 4.
Effect of temperature on cleavage of the
T197S mutant precursor protein. The purified His-tagged protein
was incubated in 10 mM Tris, pH 8.0, at different
temperatures and analyzed by SDS-PAGE.
subunit was detected by autoradiography (Fig.
5) thus showing that the acetyl group is
linked to this subunit to form the acetylated intermediate protein.
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Fig. 5.
Detection of an acetylated protein in the
processed wild-type OATase precursor. The purified His-tagged
enzyme was incubated with 14C-labeled
N-acetyl-L-glutamic acid. A,
electrophoresis in a 12% SDS-polyacrylamide gel; B,
autoradiography of the gel (exposition for 3 weeks). M,
molecular mass markers; BS OATase, B. stearothermophilus OATase.
Catalytic formation of coenzyme A by B. stearothermophilus OATase in
the presence and absence of L-glutamate
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and
subunits, which together constitute the active enzyme (2). In agreement with this hypothesis the
results of the present study show that two
and
subunits can be
formed in vitro in the presence of protease inhibitors, that
cleavage of the precursor protein occurs in a wide range of pH values
at 69 °C (a temperature at which a majority of the E. coli host proteases are inactive), and that cleavage either does
not occur or proceeds very slowly when the conserved threonine is
substituted. Therefore we conclude that the single chain precursor protein of B. stearothermophilus OATase indeed undergoes
self-catalyzed autoprocessing. Furthermore our data emphasize that the
invariant Thr-197 plays a crucial role in this autoproteolysis because
no cleavage could be detected when threonine had been replaced by glycine and because no protein assembling could be observed when the
two subunits had been expressed separately and then mixed together.
However, the hydroxyl group of serine or the thiol group of cysteine at
position 197 in the precursor protein still permits a low rate
intramolecular cleavage of mutant precursor proteins as compared with
wild-type OATase. Thus, as found for other autoprocessing pathways (see
Ref 10) it appears that the side chain of Thr, Ser, or Cys in the
OATase precursor protein can participate in proton transfer to provide
a transition from the preexisting peptide bond to an active (thiol)
ester bond which is then broken to liberate the
and
subunits,
exposing alanine at the C terminus of the
subunit and one of the
above mentioned amino acids at the N terminus of the
subunit.
Therefore, the invariant threonine, being conserved in more than 40 available OATase sequences from bacteria, archaea, and eucaryotes,
should play a crucial role in the preOATase protein self-catalyzed cleavage.
-carbonyl group of Ala-196 can then lead to the formation of a
cyclic tetrahydral intermediate with a negatively charged carbonyl
oxygen and then to the formation of a reactive ester bond. Hydrolysis
by a nucleophilic water molecule (or by another nucleophile) can lead
to the formation of the next intermediary tetrahydral state from which
a breakage reaction liberates two nascent subunits thus exposing the
threonine residue at the N terminus of the
subunit.
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Fig. 6.
Proposed mechanism of self-catalyzed
intramolecular cleavage of the OATase precursor protein.
B indicates a general base, and BH indicates a
protonated form; and
indicate OATase subunits.
The ping-pong kinetic mechanism of OATase indicates that an acetylated
intermediate is formed by covalent binding of the acetyl group from a
corresponding donor (2). This intermediate appears to be rather
unstable because, in the absence of an acetyl group acceptor, some CoA
is formed (see Table I) presumably by hydrolysis of the acetylated
intermediate. The acetylation reaction itself requires deprotonation of
a particular amino acid which should be located in the subunit as
only this subunit has been found to be acetylated in OATase. Our data
show that self-catalyzed precursor cleavage is a necessary step to form
active OATase, probably by directing appropriate folding and/or
topological organization of the active site in the oligomeric molecule.
Indeed, replacement of the invariant threonine by glycine completely
abolishes enzymatic activity. The two other substitutions, T197S and
T197C, slow down precursor protein autocleavage but also cause
significant loss of OATase activity. The fact that the T197S and T197C
mutant proteins still exhibit low enzymatic activity after complete
cleavage of precursors and association of subunits into
heterotetrameric molecules indicates that, in addition to
autoproteolysis, these mutants are affected in catalysis as well. A
probability is that an N-terminally positioned Thr-197 in the
subunit is involved in the enzymatic mechanism; this could be by
mediating the transfer of the acetyl group from the acetylated
intermediate to the L-glutamate acceptor molecule or by
inducing an appropriate conformation at the active site.
It is worth noting that this hypothesis of a crucial function for the
invariant threonine in both preprotein cleavage and OATase catalytic
mechanism could explain an apparent discrepancy between our results and
the conclusions of Abadjieva et al. (5) concerning the
maturation of active yeast OATase. These authors independently proposed
OATase autoproteolytic processing and found that the replacement of
threonine 215 (referred to by us as Thr-197 for B. stearothermophilus) by alanine abolishes activity in yeast OATase
(5). However, they also observed that a S. cerevisiae strain
deleted for the OATase gene and carrying two plasmids with separately
cloned and expressed yeast and
subunits (in the latter case a
methionine residue had been introduced at the N terminus to initiate
translation) still exhibited a weak OATase activity. In contrast, from
a plasmid providing concomitant but independent expression of B. stearothermophilus OATase
and
subunits from the same
mRNA, we have observed no complementation of E. coli
argE or argA mutants and could detect no OATase
activity whether the invariant threonine was immediately preceded or
replaced by a methionine residue. The fact that OATase activity is only partly restored when using the yeast constructions was attributed by
Abadjieva et al. to the presence of the methionine residue introduced at the N terminus of the
subunit (5). However, if we
consider that the invariant threonine is essential for the OATase-mediated catalytic mechanism and not only for processing, then
the low activity measured in yeast could be due to partial removal of
the N-terminal methionine (21); this would expose the threonine residue
at the N terminus and thus mimic the result of self-catalyzed
processing from a precursor.
Microbial OATases belong to the N-terminal transferases family of enzymes. N-acetyltransferases play important roles in various processes such as the expression of eucaryotic genes, the inactivation or activation of drugs in bacterial and mammalian cells, the morphogenesis of membranes, the formation of nodules during the establishment of symbiotic relationships between bacteria and plants, and the emergence of metabolic pathways for several amino acids. All these enzymes catalyze acetyl group transfer from acetyl-CoA to a primary amino group in different target molecules, including proteins, lipids, amino acids, sugars, and still other compounds (for a review see Ref. 23). Three-dimensional protein structures have been solved for eucaryotic HAT1 histone N-acetyltransferase (24), the GCN5 transcriptional regulator (25), both of which use histones as substrates, as well as for aminoglycoside 3-N-acetyltransferase (26), serotonin N-acetyltransferase (27), and arylamine N-acetyltransferase (28), all of which use non-proteinic substrates. Some N-acetyltransferases share a similar structural core that allows to group them in a common superfamily (29). However, in addition to self-catalyzed intramolecular cleavage OATases exhibit other features not yet detected among the above mentioned N-acetyltransferases; monofunctional OATases are able to accept the acetyl group from a donor other than acetyl-CoA, namely from N-acetyl-L-ornithine, whereas bifunctional OATases are active toward both acetyl-CoA and N-acetyl-L-ornithine. These features as well as the absence of similarity between well characterized N-acetyltransferases and OATases reflect their distant evolutionary relationship (30). Further structural and enzymatic studies of OATases may help to understand the molecular mechanism which couple self-catalyzed cleavage of the precursor to transacetylation catalysis and to elucidate the phylogenetic position of these enzymes among other N-acetyltransferases.
Acknowledgements--
We are grateful to Daniel Gigot for
providing labeled N-acetyl-L-glutamate.
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FOOTNOTES |
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* This work was supported by a grant from the Région des Pays de la Loire (Contrat de Plan Etat-Région) and by a Tournesol Program (Collaborations Franco-Belges, Communauté Flamande) for mutual visits.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
33-2-51-12-56-20; Fax: 33-2-51-12-56-37; E-mail:
Vehary.Sakanyan@chimbio.univ-nantes.fr.
Published, JBC Papers in Press, April 24, 2001, DOI 10.1074/jbc.M100392200
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ABBREVIATIONS |
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The abbreviations used are: OATase, ornithine acetyltransferase; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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