The N -acetylglutamate synthase/ N -acetylglutamate kinasefeedback regulation of the first two steps in arginine biosynthesis Katia Pauwels, Agnes Abadjieva, Pierre Hilven, Anna Stankiew
Trang 1The N -acetylglutamate synthase/ N -acetylglutamate kinase
feedback regulation of the first two steps in arginine biosynthesis Katia Pauwels, Agnes Abadjieva, Pierre Hilven, Anna Stankiewicz and Marjolaine Crabeel
Department of Genetics and Microbiology of the Vrije Universiteit Brussel, Brussels, Belgium
In Saccharomyces cerevisiae, which uses the nonlinear
pathway of arginine biosynthesis, the first two enzymes,
N-acetylglutamate synthase (NAGS) and N-acetylglutamate
kinase (NAGK), are controlled by feedback inhibition We
have previously shown that NAGS and NAGK associate in
a complex, essential to synthase activity and protein level
[Abadjieva, A., Pauwels, K., Hilven, P & Crabeel, M (2001)
J.Biol.Chem.276, 42869–42880]
The NAGKs of ascomycetes possess, in addition to the
catalytic domain that is shared by all other NAGKs and
whose structure has been determined, a C-terminal domain
of unknown function and structure Exploring the role of
these two domains in the synthase/kinase interaction, we
demonstrate that the ascomycete-specific domain is required
to maintain synthase activity and protein level
Previous results had suggested a participation of the third
enzyme of the pathway, N-acetylglutamylphosphate
reduc-tase, in the metabolon Here, genetic analyses conducted in
yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level
Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent First, the kinase becomes less sensitive to arginine feedbackinhibition in the absence of the synthase Second, and as in Neurospora crassa, in a yeast kinase mutant resistant to arginine feedbackinhibition, the synthase becomes feedbackresistant concomitantly
We conclude that the NAGS/NAGK metabolon pro-motes the co-ordination of the catalytic activities and feed-backregulation of the first two, flux controlling, enzymes of the arginine pathway
Keywords: yeast; N-acetylglutamate synthase; N-acetylglu-tamate kinase; metabolon; co-ordinated feedback inhibition
De novoarginine biosynthesis in plants and microorganisms
occurs in eight biochemical steps starting from glutamate In
the fifth step of this pathway ornithine is generated from
N-acetylornithine Two different ornithine synthesis
reac-tions can be distinguished In the linear pathway, ornithine
is generated through the hydrolysis of N-acetylornithine In
the cyclic pathway, the acetyl group of N-acetylornithine is
transferred to glutamate, thereby regenerating
N-acetylglu-tamate (Fig 1) Because it avoids the acetyl-CoA
consu-ming initial step, catalysed by N-acetylglutamate synthase
(NAGS) (EC 2.3.1.1), the cyclic pathway is energetically
more favourable However, an organism, which regenerates
N-acetylglutamate through ornithine synthesis, still requires the synthase in order to ensure a constant level of acetylated compounds during cell growth Therefore an anaplerotic role is attributed to acetylglutamate synthase in organisms using the cyclic pathway of ornithine synthesis [1,2] The linear pattern of ornithine synthesis is found in Escherichia coliand some other bacteria and archea [1–5] The cyclic pattern is more widespread among the procary-otes [6–13], and it is observed in all investigated ascomyce-tes, including Candida utilis [14], Saccharomyces cerevisiae [15], Neurospora crassa [2], and in Chlamydomonas algae [16] In the fungi, ornithine synthesis proceeds entirely in the mitochondria [17,18]
Control of the metabolic flux through a biosynthetic pathway usually occurs at the level of the first committed step and is often mediated by the end product of the pathway This classical mechanism operates in organisms using the linear pathway of arginine synthesis: arginine exerts feedbackinhibition on N-acetylglutamate synthase in E.coli and Salmonella typhimurium [19–21] In pathways where acetylglutamate is regenerated, the second enzyme of arginine biosynthesis, N-acetylglutamate kinase (NAGK) (EC 2.7.2.8) becomes the main controlling step Feedback inhibition of the kinase by arginine has been demonstrated
in several bacteria [7,22,23] Yet, metabolic control should occur on the production of acetylglutamate, regardless of its origin Therefore, feedbackinhibition on both the synthase and the kinase is believed to be general for organisms using
Correspondence to M Crabeel, Department of Genetics and
Microbiology of the Vrije Universiteit Brussel, c/o CERIA-COOVI,
Emile Gryson avenue 1, B-1070 Brussels, Belgium.
Fax: + 32 2 526 72 73, Tel.: + 32 2 526 72 84,
E-mail: mcrabeel@vub.ac.be
Abbreviations: NAGS, N-acetylglutamate synthase; NAGK,
N-acetylglutamate kinase; NAGPR, N-acetylglutamylphosphate
reductase; CD, catalytic active domain; ASD, ascomycetes specific
domain.
Enzymes: N-acetylglutamate synthase (EC 2.3.1.1), N-acetylglutamate
kinase (EC 2.7.2.8), N-acetylglutamylphosphate reductase
(EC 1.2.1.38).
(Received 25 November 2002, revised 14 January 2003,
accepted 22 January 2003)
Trang 2cyclic ornithine synthesis The feedbackregulation of these first two steps in the arginine pathway has been clearly demonstrated in the bacterium Pseudomonas aeruginosa and
in two ascomycetes: S.cerevisiae and N.crassa [24–28]
In the latter two organisms, the control of the first two steps of the arginine pathway includes an extra level of complexity Beside its own structural gene (ARG2), N-acetylglutamate synthase activity also requires the yeast ARG5,6gene (arg-6 in N.crassa) The ARG5,6 and arg-6 genes encode each a polyprotein precursor which is maturated in the mitochondrial matrix to N-acetylglu-tamate kinase and N-acetylglutamylphosphate reductase (NAGPR) (EC 1.2.1.38), catalysing, respectively, the sec-ond and third step of arginine biosynthesis [29,30] This requirement of an extra gene for the synthase activity was first observed in N.crassa, where cells containing some nonsense mutants of the arg-6 gene displayed no detectable synthase activity, despite the presence of an intact synthase encoding gene (arg-14) [28,31] An interaction between the synthase and the kinase of N.crassa was demonstrated by the yeast two-hybrid system (R L Weiss, S K Chae,
J Chung, C McKinstry, M Karaman and G Turner, University of California, Los Angeles, CA, USA, personal communication) Similar data in yeast were independently obtained by our group [32] An increase in synthase activity, expected to result from higher copy numbers of its structural gene ARG2, has only been observed with a parallel increase
in the ARG5,6 gene copy number The yeast synthase/kinase interaction was demonstrated by coimmunoprecipitation methods [32]
The physical participation of reductase, the second mat-urated gene product of ARG5,6, to the synthase/kinase com-plex, has not been proven so far Hence, it is not clear whether synthase activity and protein level require reductase How-ever, the existence of mutations in the reductase-encoding domain of the N.crassa arg-6 gene, which affect synthase activity, suggests a possible role for the reductase [28,31] Moreover, increasing the copy-number of a synthetic gene, only encoding the kinase domain of S.cerevisiae ARG5,6 gene, is not sufficient to increase the activity of yeast NAGS when coexpressed with high copy-number of ARG2 [32] Another remarkable result, concerning the regulation of the first enzymes of the arginine pathway, has been reported
by the team of R L Weiss A series of ornithine-over-producing N.crassa mutants [33], were mapped to the N-terminus of N-acetylglutamate kinase and shown to bear F81L modifications The data suggest that this single amino-acid modification of the kinase might result in the deregulation of the first two enzyme activities of the arginine pathway, leading to the hypothesis of a co-ordinated feedbackcontrol (R L Weiss, S K Chae, J Chung,
C McKinstry, M Karaman and G Turner, University of California, Los Angeles, CA, USA, personal communi-cation)
The co-ordinated regulation of the first two enzymes of the arginine pathway in ascomycetes seems to correlate with some particular features of both the synthase and the kinase genes The ascomycete N-acetylglutamate synthase enco-ding genes are conserved and appear evolutionarily not related to the gene family encoding N-acetylglutamate synthase in bacteria [32,34] Recently, the murine and the human genes encoding N-acetylglutamate synthase were
Fig 1 Simplified scheme of the arginine biosynthesis pathway in
S cerevisiae Step 1 is catalysed by N-acetylglutamate synthase
(synthase), step 2 by N-acetylglutamate kinase (kinase), step 3 by
N-acetylglutamylphosphate reductase (reductase), and step 5 by
N-acetylornithine-glutamate acetyltransferase (acetyltransferase).
Trang 3characterized and shown to pertain to the same family as the
ascomycete synthase [35,36] This apparent dual origin
of the synthases is in sharp contrast with the common
evolutionary relationship ascribed to all other genes
involved in the arginine biosynthesis in different organisms
Amino-acid sequence alignments of known members of
the N-acetylglutamate kinase family illustrate conservation
over all three domains of living organisms (Bacteria,
Archaea, and Eucarya) of a region corresponding to the
E.coli NAGK, the only NAGK of known 3D structure
[37], representing the catalytic NAGK domain However,
all the ascomycete N-acetylglutamate kinases characterized
to date, namely those of S.cerevisiae, Schizosaccharomyces
pombe, N.crassa, and Candida albicans, have two specific
features: (a) they are encoded together with NAGPR as a
bi-functional precursor protein that is processed into two
distinct enzymes in the mitochondria, and (b) they possess
an extra region of about 200 amino acids at their
C-terminus, that we call the ascomycete-specific domain
(ASD) [29,30] It is tempting to speculate that the
ascomycete-specific domain (ASD) of the kinase might
play a role in formation of the synthase/kinase protein
complex
This workinvestigates three important unsolved
ques-tions related to the structure and function of the yeast
NAGS/NAGK metabolon We analyse (a) the role of the
reductase in the activity and protein level of the synthase, (b)
the role of the ASD of the kinase in its interaction with the
synthase, and (c) the significance of the yeast NAGS/
NAGK metabolon in terms of its co-ordinated feedback
regulation by arginine
Experimental procedures
Strains and growth conditions
S.cerevisiae.The wild-type strain of this laboratory is
S1278b (Mat a) MG471 (Mat a, ura3–471) was directly
derived from S1278b by M Grenson, Universiteit
Brussel, Belgium The strains YeBR5 (Mat a, ura3–471,
Darg5::genR), YeBR6 (Mat a, ura3–471, Darg6::genR,
arg5–), and 14S31b (Mat a, ura3–, his3–) have been
described previously [32] The construction of strain SS1
(Mat a, ura3–471, Darg3), derived from MG471, has been
described [38] Strain KA44 (Mat a, ura3–, his3–, Darg2::
genR) and strain KA42 (Mat a, ura3–, his3–, Darg5,6::genR)
are derived from 14S31b and were constructed using A
Wach’s method [39]: the genomic ARG2 ORF (KA44) or
the genomic ARG5,6 ORF (KA42) were replaced by the
kanMX4 cassette and the strains were selected on the basis
of their geneticin resistance PCR analysis confirmed the
presence of the expected modification in those strains SA2,
derived from MG471, was constructed using the delitto
perfetto system developed by Storici et al [40] The
procedure allowed scarless removal of the NAGK encoding
ARG6region from the chromosomal ARG5,6 gene (deletion
from amino acid 84–493 in the ORF encoding the kinase/
reductase precursor) The resulting ura3–, Darg6 mutant
strain can be restored to prototrophy by plasmid pYB7,
expressing ARG6 from a GAL promoter This confirms
that, as expected, SA2 expresses active NAGPR from the
remaining ARG5 region of the ARG5,6 gene
All yeast strains were grown at 30C on M.am medium,
a minimal medium containing 0.02M (NH4)2SO4, 3% glucose, vitamins, and trace minerals [41] Where required, uracil,L-histidine orL-arginine was added to a concentra-tion of 25 lgÆmL)1 Genes which are transcriptionally controlled by the GAL promoter were induced by growing cells on M.gal medium (containing 2% galactose as the carbon source) instead of the usual M.am medium (containing 3% glucose) Arginine starved cells were initially grown on medium containing arginine, centrifuged, washed with water and resuspended in M.am medium without arginine Cells were starved for 3 h before harvesting them
E.coli.Strains XA4(argA–) and XC33(argC–) from the laboratory of S Baumberg have been described previously [32] Rosetta(DE3)(pRARE) is a commercial strain (Nov-agen) in which the pRARE plasmid over-expresses tRNAs for most rare E.coli codons
E.coli strains were grown at 37C on rich medium supplemented with ampicillin (25 lgÆmL)1) and chloram-phenicol (35 lgÆmL)1) where required Cell cultures at a
D600of 0.600 were induced by addition of IPTG (2,5 mM) and overnight incubation at 30C
Culture conditions for the spot tests: approximately 2 mL
of cells at D600 of 0.250 grown on rich medium plus ampicilline, were harvested by centrifugation, washed and resuspended in minimal medium to a concentration of
1010cellsÆmL)1 Drops of 10 lL of 10-fold serial dilutions (from 1010cellsÆmL)1 to 105cellsÆmL)1) were spotted on minimal medium with or without arginine (100 lgÆmL)1), and with or without IPTG (1 mM) Sets of four plates were incubated at 37C, 30 C or 25 C
Oligonucleotides BY4, BY5: [32], HP72¼ GTCTCACAACAACAATTGG CTGTGATCAAGGTG HP73¼ CACCTTGATCACA GCTAATTGTTGTTGTGAGAC HP79 ¼ CACACG ACTTCACAAAATTTTCAACTAATTTGTAACCTCT CCTGATCATAG HP80¼ CTATGATCAGGAGAGG TTACAAATTAGTTGAAAATTTTGTGAAGTCGTG GTG HP81 ¼ CACTAATTTGTAACCTCTCCTGAT AACCTCTCTTTTTGTGCTGATATTG HP82¼ CAA TATCAGCACAAAAAGAGAGGTTATCAGGAGAG GTTACAAATTAGTG AA29¼ CGTCAGACCATGG GGTGGAGGAGAATATTCGCGCATGAACTCAAG K1 ¼ GGCCATGGTTTCATCTACTAACGGCTTTT CAG K2 ¼ GGCCAAGCTTTCAACTACTTGCTGA TGAGTTGAGGGTAG K4¼ GGCCTGCAGCTCAA GGCGCACTCCCGTTCTG K8¼ GGCCTGCAGTCA ATGATGATGATGATGATGTGAAATATTTTTTTCA TTTTCCCAAC K10 ¼ CCGGAAGCTTTCAGACAC CAATAATTTTATTTTCAGGG K12 ¼ CCGGAAG CTTGTGAGCGGATAACAATTTCACACAGGAAAC AGACCATGCCTCGTCCCGAGGGAGTTAACACC Plasmid constructs
Table 1 gives an overview of the main features of the plasmids used in this work, including the new constructs Plasmids pHP17, pHP21, and pHP22 (expressing the
Trang 4ARG5,6gene altered in its NAGK-encoding ARG6 region)
were all constructed by recombinant PCR, usingS1278b
genomic DNA as template Two overlapping fragments
were generated in a first PCR amplification step, then
self-annealed, elongated to duplex DNA, and amplified in a
second PCR step using the two external oligonucleotide
primers of the two oligonucleotide pairs of the first PCRs
These external primers are designed to add adequate
restriction sites for classical cloning in the pYX223 vector
(from R&D systems) The latter is a 2 micron-based yeast–
E.colishuttle vector, bearing HIS3 as selection marker, and
in which the expression of the inserted genes is put under the
control of a GAL promoter The BY4/HP73 and HP72/
BY5 primer pairs were used to construct pHP17, BY4/
HP79, and HP80/BY5 for pHP21 and BY4/HP81 and
HP82/BY5 for pHP22
Plasmids of the pYK series were all derived from the
E.coliexpression vector pTrc99a (Pharmacia) and contain
different insertions, all obtained by PCR amplification The
inserted fragments allow the expression of the ORF under
the transcriptional control of the IPTG-inducible strong
bacterial trp-lac promoter and under the translational
control of an appropriate Shine–Dalgarno sequence
Plasmids pYK1 expresses the ARG6 ORF, cloned as an
NcoI–HindIII fragment amplified using K1 and K2 as
primers and plasmid pYB3 as a template Plasmid pYK7
expresses the ARG2 ORF, cloned as a NcoI–PstI fragment
(primers AA29 and K8 and pYB2 as a template) With
primer AA29, a tag of six histidine codons is fused in frame
to the C-terminus of the ARG2 ORF for immunodetection
of the enzyme Plasmid pYK8 was obtained by inserting
the ARG6 ORF and its trp-lac promoter (from position
)115) as a PstI–HindIII fragment (primers K4/K2,
tem-plate pYK1) into plasmid pYK7 The artificial operon of plasmid pYK11 expresses a bi-cistronic ARG5/ARG6 mRNA under the control of the trp-lac promoter and was obtained by inserting a HindIII fragment (primers K10/K12, template pYK3), containing the reductase encoding region, into plasmid pYK8 Primer K12 has a 35-nucleotide 5¢ extension containing a ribosome site and
an initiator codon
DNA sequencing The nucleotide sequence of the ARG5,6 gene, cloned in the plasmids pHP17, pHP21, pHP22 and pYK11, was determined Beside the intended modification or deletion, these constructions, issued from independent PCR-amplifications, share additionally the same 15 single-nucleotide differences with respect to the data base sequence These S1278b-specific differences with respect
to the ARG5,6 gene of strain S288c, used to establish the data base, result in only one amino-acid difference: the E803K modification in the region of the gene encoding NAGPR
Enzyme activity assays Acetylglutamate synthase This enzyme activity was meas-ured by a radioassay usingL-[U-14C] glutamate and acetyl-CoA as substrates, as described previously [32] Dependent
on the experiment, 400 mL to 2 L of yeast cultures at
D600 0.4 were required Extracts were prepared using the French press For E.coli experiments, cells of 100 mL cultures (induced overnight) were collected and extracts were obtained by ultrasonication
Table 1 Main features of the plasmids used in this work.
Plasmids Cloning vector
Origin of
pYB2 pYX213 (2l, URA3) S1278b PromoterGAL ARG2 ORF-HAtag (32) WT NAGS-HA
pYB3 pYX223 (2l, HIS3) S1278b PromoterGAL ARG5,6 ORF (32) WT NAGK + WT NAGPR
(amino acids 1–863) pYB7 pYX223 (2l, HIS3) S1278b PromoterGAL ARG6 (32) WT NAGK (amino acids 1–537) pYB8 pYX223 (2l, HIS3) S1278b PromoterGAL ARG5 (32) WT NAGPR (amino acids 1–38 +
amino acids 494–863) pHP17 pYX223 (2l, HIS3) S1278b PromoterGAL ARG5,6 ORF (F99L) FBRNAGK + WT NAGPR
pHP21 pYX223 (2l, HIS3) S1278b PromoterGAL ARG5,6 ORF
(Damino acids 355–493)
NAGK (DASD) + WT NAGPR pHP22 pYX223 (2l, HIS3) S1278b PromoterGAL ARG5,6 ORF
(Damino acids 85–347)
NAGK (DCD) + WT NAGPR p238 YCp50 (ARS-CEN, S288c GCN4 (4 uORFs untranslated) a Constitutive expression of Gcn4p
URA3)
pYK8 pTrc99a S1278b PromoterTrc ARG2 ORF-HIS6tag +
PromoterTrc ARG6
WT NAGS-HIS6 + WT NAGK (amino acids 58–513)
pYK11 pTrc99a S1278b PromoterTrc ARG2 ORF-HIS6tag +
PromoterTrc ARG6 + ARG5 operon
WT NAGS-HIS6 + WT NAGK (amino acids 58–513) + WT NAGPR (amino acids 531–863)
a Gift of A Hinnebusch, National Institute of Child Health and Human Development, Bethesda, MD, USA.
Trang 5Acetylglutamate kinase The assay used to measure
NAGK activity has been described previously [18] In total
yeast extracts, this assay detects two distinct enzymatic
reactions [18,26] As the interfering activity is not inhibited
by arginine (in contrast to the full inhibition of NAGK), a
blankincluding 5 mMarginine was used by Jauniaux et al
to subtract the interfering activity [18] Because we used
arginine feedbackresistant mutants in this work, we used
adapted blanks containing 50 mM arginine In some
experiments, the blanks were reaction mixtures incubated
without the substrate acetylglutamate This explains the
presence of a residual activity, resistant to arginine
inhibi-tion, in Fig 5 (about 15% of the initial kinase activity) A
kinase activity similar to that residual activity is measured in
extracts of strain KA42 bearing a full deletion of the
ARG5,6gene
All NAGS and NAGK activities reported in this work
are means of at least three independent experiments
Standard deviations generally did not exceed 15%
Western blots
A standard chemiluminescence Western blotting protocol
(Roche) was used to analyse the yeast NAGS expressed in
E.coli from plasmids pYK7, pYK8, and pYK11 Equal
amounts of total proteins of the different crude extracts were
separated by SDS/PAGE on 12% gels, and then blotted on
an ECL Hybond nitrocellulose membrane (Amersham
Pharmacia Biotech) in transfer buffer [25 mM Tris,
192 mMglycine, 20% (v/v) methanol] using a Mini
PRO-TEAN 3 blotting cell (Bio-Rad) Specific primary mouse
anti-HIS Ig (Santa Cruz Biotechnology) (0.1 ngÆmL)1) and
40 UÆmL)1peroxidase-labelled secondary antibody (Roche)
were used to detect the tagged synthase protein
Chemilu-minescence was monitored by autoradiography Detection
of Haemaglutinin (HA)-tagged NAGS, expressed by the
pYB2 plasmid in yeast cells, was as described previously [32]
Results
At physiological levels, the presence
of N-acetylglutamyl phosphate reductase
is dispensable to synthase activity
In order to determine the influence of N-acetylglutamyl
phosphate reductase on the activity of N-acetylglutamate
synthase, the synthase activity was measured in different mutants carrying deletions in relevant parts of the chromo-somal ARG5,6 gene Strain YeBR6 expresses neither the kinase nor the reductase, while only the kinase is expressed
by strain YeBR5 [32] A new strain, SA2 bears a deletion of the kinase-encoding domain of ARG5,6 and has its remaining reductase-encoding domain fused to the mito-chondrial targeting peptide The SS1 strain is used as the ARG5,6+ positive control SS1 bears an ARG3 deletion rendering the control strain arginine-dependent, like the tested strains SS1, SA2, YeBR5 and YeBR6 are all directly derived from MG471
In a crude extract of wild-type yeast, the physiological level of synthase activity is barely detectable The detection becomes even more difficult for the strains requiring arginine for cell growth, presumably due to a tight binding
of the feedbackinhibitor Moreover, adequate removal of the inhibiting arginine, by dialysis or repeated gel filtration,
is limited by the lackof stability of the synthase To overcome this difficulty, we choose to assay NAGS in extracts of arginine-starved cells (see strains and growth conditions) The arginine deprivation results in a Gcn4p-mediated transcriptional activation of the ARG2 gene (K Pauwels and M Crabeel, unpublished results) and reduces the pool of the feedbackinhibitor Even higher levels of synthase activity were detected in strains bearing the p238 plasmid, due to a constitutive production of the Gcn4p transcriptional transactivator (Table 2)
Synthase activity was assayed in crude extracts of arginine starved SS1, YeBR5, SA2 and YeBR6, with and without the plasmid p238 (Table 2) No synthase activity was detectable in absence of the kinase (SA2 and YeBR6 vs SS1) In contrast, the absence of reductase did not affect considerably the synthase activity, though a small decrease was observed (YeBR5 vs SS1) These data demonstrate that, at physiological level, the synthase activity requires the presence of the kinase, and that the additional presence of the reductase is dispensable
Activity and protein level of the yeast synthase expressed inE coli, require the coexpression
of the yeast kinase but not of the yeast reductase The E.coli strain XA4 (argA–), which is defective in N-acetylglutamate synthase, cannot be restored to arginine prototrophy by a trp-lac-promoter-driven expression of the
Table 2 Physiological levels of the N-acetylglutamate synthase in strains bearing different deletions in the ARG5,6 gene.
Status of
NAGS activity (nmolÆmin)1Æmg)1protein)
a Plasmid p238 expresses Gcn4p constitutively; b below detection.
Trang 6yeast synthase [32] In the present study we analysed the
influence of the additional expression of the yeast kinase,
and of the yeast kinase and reductase together Three
plasmids, derived from pTrc99A, are designed to express
yeast synthase (pYK7), yeast synthase and kinase (pYK8)
or yeast synthase, kinase and reductase (pYK11) These new
constructions, including the empty vector pTrc99a, are
transformed in strain XA4 SDS/PAGE/Coomassie Blue
analysis and kinase activity assays confirmed that XA4
(pYK8) and XA4(pYK11) are over-expressing functional
kinase protein (data not shown) Beside the kinase protein,
XA4 (pYK11) expresses the reductase protein, however, in
lower amounts The functionality of the reductase, encoded
by pYK11, was verified by complementation of the
reductase deficient E.coli strain XC33 (argC–) (data not
shown)
First, all four plasmids were tested for their efficiency to
complement the argA–deficiency of the XA4 strain, using
spot tests of serial dilutions incubated at 37C Under
noninducing conditions (Fig 2A), pYK8 and pYK11 (both
expressing the kinase protein) allow growth of the
arginine-deficient mutant in the absence of arginine On the other
hand, plasmids pYK7 and the empty vector pTrc99a (both
lacking the yeast kinase ORF) were completely unable to
complement the mutation These data demonstrate that the
presence of the kinase is essential to yeast synthase activity
while the additional presence of the reductase (pYK11 vs
pYK8) does not improve complementation The
observa-tion that complementaobserva-tion is even slightly lower in the
presence of the reductase, could be due to a lower copy
number of pYK11, which is larger than pYK8
Unexpect-edly, expression of pYK8 and pYK11 under induced
conditions did not improve the efficiency of
complementa-tion of the argA– XA4 strain (Fig 2C) In contrast, it
appeared to be toxic to the cell This cell toxicity was
demonstrated by the severe growth handicap observed
when ITPG and arginine were supplemented together to the
medium (Fig 2D vs 2C) On the other hand, the absence of
growth of strain XA4(pYK7) under inducing conditions
(Fig 2C) demonstrates the incapacity of the plasmid that bears only the synthase gene to complement the arginine deficiency, rather than revealing cell toxicity This is shown
by a similar behaviour in growth of XA4(pTrc99A) and XA4(pYK7), in all conditions used
Same series of spot test were also realized with plate incubations at 30C and 25 C These milder temperatures did not allow any growth of XA4(pYK7) in the absence of arginine, but complementation of the synthase deficiency by pYK8 and pYK11 slightly and gradually improved with decreasing incubation temperatures (data not shown)
In a second step, synthase specific activity was determined
in XA4 strains bearing one of the four plasmids mentioned above, following an overnight induction at 30C with 2.5 mMIPTG Table 3 summarizes the results No synthase activity was detected for XA4(pYK7) (expressing only the yeast synthase) and high activity was measured for XA4(pYK8) (coexpressing synthase and kinase) Compared
to XA4(pYK8), XA4(pYK11), which additionally expresses the reductase, showed a slight decrease in synthase activity, which can presumably be ascribed to a lower plasmid copy number
To test whether the absence of synthase activity is the result of low levels of NAGS protein, an immunoWestern
Table 3 Yeast N-acetylglutamate synthase specific activity in the XA4 (argA – ) E coli background.
Plasmid
Yeast enzymes expressed
NAGS activity after IPTG induction (nmolÆmin)1Æmg)1 protein)
pYK11 NAGS + NAGK + NAGPR 30
a
Below detection.
Fig 2 Spot growth tests of the E coli strain XA4(argA–) transformed with various plasmids as indicated In each row, from left to right, 10 lL of 10-fold serial dilutions of a cell suspension (going from 10 10 cellsÆmL)1to 10 5 cellsÆmL)1) were spotted, either under noninducing conditions, without arginine (A) and with arginine (B); or under inducing conditions, without arginine (C) and with arginine (D) Plates were incubated at 37 C.
Trang 7blot analysis was performed, comparing equal amounts of
total proteins from crude extracts of the four type of
transformants (Fig 3) The synthase protein was detected
by its His6 tag in extracts of the strains bearing pYK8 or
pYK11, but not in extracts of a strain bearing pYK7 A
small difference in protein concentration was observed
between pYK8 and pYK11 in some blots, ascribed to a
lower plasmid copy number Thus, protein concentration
data correspond to the data of the growth assays and of the
activity measurements Therefore, unless kinase is
coex-pressed, yeast synthase appears unstable, both in a
hetero-logous bacterial background (present data) and in an yeast
homologous context [32] This suggests an intrinsic
insta-bility of the synthase protein Furthermore, in a
hetero-logous bacterial background, the supplementary presence of
the yeast reductase, in addition to the yeast kinase, does not
enhance synthase activity or levels
The ascomycete-specific domain ofN-acetylglutamate
kinase is required to maintainN-acetylglutamate
synthase activity and protein level
Yeast N-acetylglutamate kinase consists of two
distinguish-able domains The N-terminal domain is conserved in both
eucaryotes and procaryotes and is therefore inferred to be
the catalytic active domain (CD) The C-terminal domain is
specific to ascomycetes (ASD) It extends from about amino
acid 348 to a residue located between amino acid 510 and
540, the region in which the kinase/reductase precursor is
maturated [29] We addressed the question whether the two
kinase domains are needed to observe synthase activity and
stability By inference, this would indicate a role for each
domain in the association of the NAGS/NAGK in a
complex
For this experiment, new high copy number plasmids
were derived from pYB3, each lacking one of the kinase
domains Plasmid pYB3 encodes the full length ARG5,6
gene, plasmid pHP21 is truncated for the ascomycete specific domain of the kinase (Daa355–493) and plasmid pHP22 is truncated for the catalytic domain of the kinase (Daa85–347) The functionality of the kinase protein encoded by those plasmids was assessed by transforming the plasmids in the Darg5,6 genetic background of strain KA42 and measuring kinase activity As expected KA42(pHP22) lacks any kinase activity while KA42(pHP21) keeps more than 50% of the wild-type kinase activity as compared to KA42(pYB3) (data not shown), implying that the ASD-truncated kinase is stably expressed
We then analysed the synthase activity and protein level when the synthase protein was coexpressed with one of the truncated kinases Therefore, pYX223, pYB3, pHP21 and pHP22 were cotransformed in 14S31b with pYB2, which is
a GAL promoter-driven plasmid, over-expressing the synthase fused to a C-terminal haemaglutinin (HA)-tag The first two combinations served as a negative and a positive control, respectively The host strain 14S31b has a ARG2, ARG5,6 genetic background circumventing the need
to add arginine in the growth medium, but explaining the low background synthase activity and protein level of the negative control
Table 4 summarizes the values of the synthase specific activity measured under galactose promoter inducing growth conditions As expected, the coexpression of wild-type kinase and synthase resulted in high synthase activity The combined expression of synthase with each of the truncated kinase proteins, however, showed no increase in the synthase activity compared to the negative control This suggests that complex formation does not occur, resulting in synthase protein instability Alternatively, a non-productive but stable association can be the cause of this inactivity To assess the synthase protein level, an immunoWestern blotting was performed on crude extracts of several strains,
as shown in Fig 4A No synthase was detectable in any of the samples, except for 14S31b(pYB2 + pYB3), which was used as a positive control Only when the gels were deliberately overloaded, did synthase become detectable in the extracts from strains bearing pHP21 and pHP22, yet in amounts comparable to the basal level produced in the negative control (Fig 4B)
The results demonstrate that the ascomycete-specific domain of the kinase is required for accumulation of the synthase However, if this domain is assumed to be
Fig 3 NAGS detection by immunoWestern blot analysis of total
pro-tein extracts of E coli strain transformed with plasmid pYK7 expressing
His 6 -tagged yeast N-acetylglutamate synthase (NAGS), pYK8
expres-sing His 6 -tagged NAGS and N-acetylglutamate kinase (NAGK) or
pYK11 expressing His 6 -tagged NAGS, NAGK and N-acetylglutamyl
phosphate reductase Plasmid pTrc99a is the corresponding empty
cloning vector MM, molecular mass markers The arrow indicates the
protein band corresponding to NAGS.
Table 4 N-acetylglutamate synthase specific activity in strains coex-pressing promoter GAL-driven ARG2 and ARG5,6 genes: effect of domain deletions in the N-acetylglutamate kinase.
Strain
Status
of NAGK NAGS activity
(nmolÆmin)1Æmg)1 protein)
CDa ASDb 14S31b (pYB2 + pYX223) – – 14
a CD, catalytic domain; b ASD, ascomycete specific domain.
Trang 8expressed and to be stable in the CD truncated kinase, these
data indicate that the ASD is insufficient to maintain the
activity of the synthase
The kinase F99L mutant leads to arginine feedback
resistance of both the kinase and the synthase
R L Weiss and coworkers found that the F81L
modifica-tion in the N-acetylglutamate kinase of N.crassa renders
the enzyme resistant to arginine feedbackinhibition
(R L Weiss, S K Chae, J Chung, C McKinstry,
M Karaman and G Turner, University of California, Los Angeles, CA, USA, personal communication) Align-ment of the amino-acid sequences of S.cerevisiae, S.pombe, C.albicans, and N.crassa kinases shows the phenylalanine
81 of N.crassa to be conserved in ascomycete kinases It corresponds to the phenylalanine 99 in S.cerevisiae We constructed the yeast kinase ARG5,6 F99L mutant in a vector with a GAL promoter, yielding plasmid pHP17 Plasmid pYB2, encoding the yeast synthase, was cotrans-formed with pHP17 in the strain YeBR6 YeBR6 (pYB2 + pYB3), over-expressing both the wild-type kinase and synthase, was used as a reference strain The trans-formants were grown on galactose medium and N-acetyl-glutamate kinase activity in cell extracts was assayed in the presence of increasing arginine concentrations Figure 5A compares the arginine inhibition curves of the wild-type and F99L mutant kinases The arginine concentration required
to inhibit 50% of the activity of the wild-type kinase (I0.5) is 0.1 mM, a value that is comparable to an I0.5of 0.05 mM
Fig 4 ImmunoWestern blot detection of N-acetylglutamate synthase in
total protein extracts of yeast strain 14S31b bearing plasmid pairs as
indicated above the lanes pYB2 expresses a haemaglutinin-tagged
NAGS, pYB3 expresses the wild-type NAGK/NAGPR, pHP21 and
pHP22 are derived from pYB3 and, respectively, lackthe
ascomycete-specific domain and the catalytic domain of the kinase encoding
region, pHP17, also derived from pYB3, bears the F99L modification
in NAGK pYX213 and pYX223 are the empty cloning vectors (A)
Equal amounts of total protein were loaded in lanes 1–4, and double
that amount in lanes 6–9 (B) All lanes contain equal amounts of total
protein (C) Lanes 1 and 2 contain 7.5 lg total protein, lanes 3 and 4
contain 15 lg, and lanes 5 and 6 contain 30 lg MM is the molecular
mass standard.
Fig 5 Feedbackinhibition by arginine of yeast N-acetylglutamate (A) kinase and (B) synthase activities in extracts of strain YeBR6 (pYB2 + pYB3) expressing NAGS, NAGK and NAGPR (d) and strain YeBR6(pYB2 + pHP17) expressing NAGS, mutant F99L NAGK and NAGPR (s), after growth on galactose medium The insert shows the effect of arginine at higher concentrations, (A) up to
100 m M , (B) up to 10 m M The arginine-resistant residual activity in A
is due to a distinct enzymatic activity not encoded by ARG6.
Trang 9mentioned by Hilger [26] It is also comparable to the I0.5of
0.075 mMdetermined for the wild-type kinase of N.crassa
[27] In the absence of arginine the kinase specific activity in
the extract from yeasts carrying the F99L mutation was
only one half of the activity of wild-type yeast It remains
susceptible to feedbackinhibition by arginine, but 100 times
higher arginine concentration is required to reach 50%
inhibition (I0.5of 10 mM)
Synthase activity and its arginine sensitivity were also
assayed using the same extracts (Fig 5B) In the presence of
the wild-type kinase, 0.015 mMarginine is required to reach
I0.5 of the synthase This value corresponds with the
I0.5-value of 0.02 mM published by Wipf and Leisinger
[25] It is noticeable that the yeast synthase is 10 times more
sensitive to feedbackinhibition by arginine than the
N.crassasynthase (50% inhibition at 0.16 mM[28]) When
coexpressed with the F99L mutant kinase, the synthase
behaves quite differently than when coexpressed with the
wild-type kinase The synthase specific activity is reduced
fivefold and, like the mutant kinase, the enzyme becomes
much less sensitive to arginine feedbackinhibition (I0.5of
0.75 mM) The reduction in activity is not a consequence of
a loss in synthase protein, as immunoWestern blots revealed
equal amounts of the haemaglutinin-tagged synthase in
both transformants (Fig 4C)
The increased resistance to feedbackinhibition of the
wild-type synthase, resulting from the presence of the
feedback-resistant F99L kinase, suggests a mechanism of
co-ordinated feedbackregulation of the synthase and the
kinase
Arginine feedback inhibition ofN-acetylglutamate
kinase is altered in the absence ofN-acetylglutamate
synthase protein
Proper feedbackinhibition of the synthase appears to
require an association with a feedback-sensitive kinase To
find out whether the opposite is also true, we studied
arginine feedbackinhibition of the kinase in the presence
and absence of the synthase protein KA44, a strain derived
from 14S31b (ura3–, his3–) and lacking the ARG2 ORF, was
constructed and cotransformed with (pYB2 + pYB3) and
(pYX213 + pYB3) Because of the growth requirement of
the latter transformants, cells were grown on galactose
medium supplemented with arginine Kinase specific
acti-vity in extracts was assayed in the presence of increasing
arginine concentrations The results are presented in Fig 6
In extracts of KA44(pYB2 + pYB3), the wild-type
kinase proved to be sensitive to arginine inhibition The
inhibition curve displays a normal hyperbolic shape and the
I0.5-value of 0.26 mMarginine in the illustrated experiment
(Fig 6) is comparable to the I0.5of 0,1 mMmeasured with
YeBR6(pYB2 + pYB3) extracts (Fig 5A) In fact, three
similar, less detailed experiments (data not shown), display
an I0.5-value closer to 0.1 mM Interestingly, the apparent
affinity of the kinase for the feedback inhibitor is markedly
lower when the synthase is absent (I0.5-value of 1.5 mM) In
addition, the inhibition curve becomes reproducibly
sigmo-idal These data show that the kinase requires an interaction
with the synthase for its normal arginine
feedbackinhibi-tion Furthermore, the values of the specific activity of the
kinase were reproducibly two times higher in extracts of
KA44(pYX213 + pYB3) compared to those of KA44 (pYB2 + pYB3), suggesting that the association with the synthase inhibits partially the kinase activity
Discussion
In our previous work, we showed that synthase forms a complex with the kinase, an association essential to synthase activity and synthase protein accumulation In contrast, no physical interaction could be demonstrated conclusively between synthase (or kinase) and reductase despite the fact that some data suggested a role of the reductase for synthase activity [32]
To investigate further the role of the reductase in the metabolon, we have now followed two new genetic approaches First, the activity of the synthase, expressed from its natural locus, was measured in yeast deletion mutants lacking relevant parts of the chromosomal ARG5,6 gene Second, synthase activities and protein accumulation were monitored by over-expressing yeast genes in the heterologous E.coli background Both approaches led to the same conclusions, namely that synthase activity is strictly dependent on the presence of the kinase and essentially independent of the reductase This dispensibility
of the reductase for synthase activity, is in contrast with the previous results obtained in a context of over-expression in yeast, which had indicated an apparent requirement of the reductase [32] One (unexplored) hypothesis that might explain the discrepancy, is that the bulkof over-expressed kinase is inefficiently targeted to the yeast mitochondria in the absence of reductase
The present data in E.coli further show that no synthase protein is detectable in the absence of kinase, a situation similar to the one observed previously in yeast We attribute this drastic reduction in steady state concentration of the protein to an instability of the yeast synthase when not associated to the kinase Because it is also observed in the heterologous E.coli background, this apparent instability is likely to be an intrinsic feature of the protein, rather than to result from of a yeast specific degradation process Alter-natively, the uncomplexed synthase might present structural features rendering it susceptible to proteolytic degradation
Fig 6 Feedbackinhibition by arginine of yeast N-acetylglutamate
k inase activity in extracts of Darg2 strain KA44(pYB2 + pYB3) (d) and KA44(pYX213 + pYB3) (s), after growth on galactose medium supplemented with arginine.
Trang 10in general In any case, the lackof synthase protein in the
absence of kinase has been observed with expression
systems using totally different promoters and translation
initiation signals Therefore, the hypothesis that it results
from an effect on transcription or translation can be
reasonably excluded
The data in hand today do not allow to tell if the lackof
synthase activity in the absence of kinase fully correlates
with the physical disappearance of the enzyme, or if inactive
free synthase can subsist However, as discussed below, the
kinetic properties of the synthase are likely to be modulated
by its association with the kinase
The role of the catalytic and the ascomycete specific
domain of NAGK in complex formation with NAGS was
tested in an indirect way Our approach is based on the
knowledge that synthase activity and protein levels are
dependent upon the enzyme association with the kinase (32
and new data above) Therefore, capacity for complex
formation was deduced from measurements of
over-expressed synthase activity and from estimations of the
concentrations of over-expressed synthase protein Present
data showed that deletions of the catalytic domain (CD) or
the ascomycete specific domain (ASD) of the kinase both
result in the loss of synthase activity and stability As the
ASD-truncated kinase is shown to be stable and active, it
implies that the ASD of the kinase is necessary for a
productive association with the synthase The presence of
CD-truncated kinase in yeast extracts could not be
demonstrated (neither over-expressed truncated kinases
nor the wild-type are detectable by SDS/PAGE/Coomassie
Blue staining analysis), but if it is assumed to be stable,
then the data show that the ASD is not sufficient for
association with the kinase Previous data revealing highly
reduced amounts of synthase when coexpressed with
N-terminally His10-tagged kinase [32], support the
hypo-thesis that the ASD of the kinase does not suffice for
complex formation
By analogy with the F81L substitution of N.crassa
kinase, which renders it feedback resistant, a new mutant of
the yeast kinase (F99L substitution) was constructed Our
results show that the yeast mutant kinase is feedback
resistant as well In comparison to the wild-type yeast
kinase, 100 times more arginine is required to reach
half-inhibition of the F99L yeast mutant kinase Our results
illustrate further that feedbackregulation of the wild-type
yeast synthase is strongly dependent upon the presence of a
normally regulated kinase In the presence of the wild-type
kinase, the synthase is fully inhibited by 0.1 mMarginine,
while 10 mMarginine is required to inhibit completely the
synthase activity when the partner is a feedback-resistant
mutant kinase Moreover, the kinetic properties of the
synthase appear dependent upon its association with the
kinase Indeed, in the context of the mutated kinase,
the synthase specific activity was reduced by 80% while the
amount of enzyme remained unchanged
Contrasting with the strict requirement of NAGK for
NAGS activity, the absence of NAGS increased the activity
of NAGK by approximately twofold, possibly reflecting
inhibition of NAGK in the NAGS/NAGK complex The
presence of NAGS had also the effect of rendering
hyperbolic the inhibition of the kinase by arginine, whereas
in absence of NAGS the inhibition was sigmoidal and
exhibited an increased I0.5-value, strongly suggesting that more than one site for arginine has to be occupied to inhibit the kinase Although the present results demon-strate quite different apparent affinities for arginine of the kinase and the synthase, the data do not allow to decide if specific inhibitory sites for arginine exist in the two enzymes or if only the kinase possesses a binding site for the inhibitor In any case, the mutual influence of each enzyme on the other concerning its susceptibility to arginine inhibition suggest the existence of either a cross-talkbetween the inhibitory sites of the two enzymes, or an intermoleculair transmission of an inhibitory signal from a binding site on the kinase to the catalytic site of the synthase Both are in agreement with the hypothesis of co-ordinated feedbackregulation of NAGS and NAGK
in yeast, as proposed initially for N.crassa (R L Weiss,
S K Chae, J Chung, C McKinstry, M Karaman and
G Turner, University of California, Los Angeles, CA, USA, personal communication)
Acknowledgements
K P is the recipient of a Specialization Grant from the IWT (Vlaams Instituut voor de bevordering van het wetenschappelijk-technologisch onderzoekin de industrie) We thankJ P Ten Have for his help with the figures and tables.
References
1 Cunin, R., Glansdorff, N., Pierard, A & Stalon, V (1986) Bio-synthesis and metabolism of arginine in bacteria Microbiol.Rev.
50, 193–225.
2 Davis, R (1986) Compartmental and regulatory mechanisms in the arginine pathways of Neurospora crassa and Saccharomyces cerevisiae Micr.Rev.50, 280–313.
3 Harris, B & Singer, M (1998) Identification and characterization
of the Myxococcus xanthus argE gene J.Bacteriol.180, 6412– 6414.
4 Van de Casteele, M., Demarez, M., Legrain, C., Glansdorff, N & Pie´rard, A (1990) Pathways of arginine biosynthesis in extreme thermophilic archaeo- and eubacteria J.Gen.Microbiol.136, 1177–1183.
5 Xu, Y., Liang, Z., Legrain, C., Ruger, H & Glansdorff, N (2000) Evolution of arginine biosynthesis in the bacterial domain: novel gene-enzyme relationships from psychrophilic Moritella strains (Vibrionaceae) and evolutionary significance of N-alpha-acetyl ornithinase J.Bacteriol.182, 1609–1615.
6 Udaka, S & Kinoshita, S (1958) Studies on 1-ornithine fermen-tation I The biosynthetic pathway of 1-ornithine in Micrococcus glutamicus J.General Appl.Microbiol.4, 272–282.
7 Hoare, D & Hoare, S (1966) Feedbackregulation of arginine biosynthesis in blue-green algae J.Bacteriol.92, 375–379.
8 Haas, D., Kurer, V & Leisinger, T (1972) N-acetylglutamate synthetase of Pseudomonas aeruginosa An assay in vitro and feedbackinhibition by arginine Eur.J.Biochem.31, 290–295.
9 Shinners, E & Catlin, B (1978) Arginine biosynthesis in Neisseria gonorrhoeae: enzymes catalyzing the formation of ornithine and citrulline J.Bacteriol.136, 131–135.
10 Meile, L & Leisinger, T (1984) Enzymes of arginine biosynthesis
in methanogenic bacteria Experientia 40, 899–900.
11 Sakanyan, V., Kochikyan, A., Mett, I., Legrain, C., Charlier, D., Pie´rard, A & Glansdorff, N (1992) A re-examination of the pathway of ornithine biosynthesis in a thermophilic and two mesophilic Bacillus species J.Gen.Microbiol.138, 125–130.