These data sug-gest strongly that the complex of Sno1p and Snz1p is a glutamine amidotransferase with the former serving as the glutaminase, although the activity was barely detectable w
Trang 1Characterization of the products of the genes SNO1 and SNZ1 involved
Yi-Xin Dong, Shinji Sueda, Jun-Ichi Nikawa and Hiroki Kondo
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Japan
Genes SNO1 and SNZ1 are Saccharomyces cerevisiae
homologues of PDX2 and PDX1 which participate in
pyri-doxine synthesis in the fungus Cercospora nicotianae In
order to clarify their function, the two genes SNO1 and
SNZ1were expressed in Escherichia coli either individually
or simultaneously and with or without a His-tag When
expressed simultaneously, the two protein products formed
a complex and showed glutaminase activity When purified
to homogeneity, the complex exhibited a specific activity of
480 nmolÆmg)1Æmin)1as glutaminase, with a Kmof 3.4 mM
for glutamine These values are comparable to those for
other glutamine amidotransferases In addition, the
gluta-minase activity was impaired by 6-diazo-5-oxo-L-norleucine
in a time- and dose-dependent manner and the enzyme was protected from deactivation by glutamine These data sug-gest strongly that the complex of Sno1p and Snz1p is a glutamine amidotransferase with the former serving as the glutaminase, although the activity was barely detectable with Sno1p alone The function of Snz1p and the amido acceptor for ammonia remain to be identified
Keywords: glutamine amidotransferase; pyridoxine biosyn-thesis; Saccharomyces cerevisiae; SNO1; SNZ1
Pyridoxal phosphate plays a crucial role in amino acid
metabolism Pyridoxine and its phosphate are the
precur-sors of pyridoxal phosphate and the major forms of vitamin
B6 Pyridoxine biosynthesis in Escherichia coli has been
studied extensively but only recently has the whole synthetic
pathway been finally established [1–3] There are organisms
such as budding yeast, Saccharomyces cerevisiae, which
also synthesize pyridoxine but in a different pathway This
notion is based in part on an observation that the nitrogen
of pyridoxine is derived from the amide group of glutamine
in yeast [4], while glutamate is the source of the ring nitrogen
in E coli [5] Recently, two independent groups identified
pyroAand SOR1 (PDX1) as participating in pyridoxine
synthesis in fungi Cercospora nicotianae and Aspergillus
nidulans, respectively [6,7] They are homologous genes and
their homologues are distributed widely in various
organ-isms, but nothing of their function is known except that
SNZ1, the yeast homologue, works in the stationary phase
of yeast cells together with SNO1 [8] In addition to these
observations, it was shown recently that a pentose or
pentulose constitutes the skeleton of pyridoxine in yeast
[9,10] Herein, we report that Sno1p and Snz1p serve as a
glutaminase to supply ammonia for the ring nitrogen of
pyridoxine in yeast Based on these and other lines of
evidence, a putative synthetic pathway to pyridoxine is
presented in the Discussion in which ribulose 5-phosphate and ammonia serve as the key starting or intermediary material
Experimental procedures
Materials Inorganic salts and common organic chemicals including amino acids, nucleic bases and vitamins were obtained from commercial sources Acetylpyridine adenine dinucleotide (APAD) and 6-diazo-5-oxo-L-norleucine (DON) were from Sigma (St Louis, MO, USA) Glutamate dehydrogenase from bovine liver was obtained from Oriental Yeast (Tokyo, Japan) Reagents for genetic engineering such as restriction enzymes were purchased from Takara (Kyoto, Japan) and New England Biolabs (Beverly, MA, USA) Oligonucleo-tides were custom synthesized by Hokkaido Science (Sap-poro, Japan) Plasmid YEpM4 was a 2 lm DNA-based shuttle vector with gene LEU2 as the selectable marker [11] Plasmids pET21a, pET21d (both ampicillin resistant), pET24a (kanamycin resistant) and His-bind columns were from Novagen (Madison, WI, USA) The TOPO TA cloning kit was the product of Invitrogen (Carlsbad, CA, USA)
Strains and media The S cerevisiae strain used in this study was D373-1 (MATa, leu2, his3, trp1) [12] The following medium was used to grow yeast: YPD [1% yeast extract, 2% polypep-tone, 2% glucose (v/v/v)] and synthetic medium [glucose,
20 g; (NH4)2SO4, 1.02 g; KH2PO4, 0.875 g; K2HPO4, 0.125 g; CaCl2ÆH2O, 0.02 g; NaCl, 0.01 g; MgSO4Æ7H2O, 0.05 g; CuSO4Æ5H2O, 40 lg; MnSO4ÆH2O, 400 lg; FeCl3Æ 6HO, 200 lg; ZnSOÆ7HO, 400 lg; NaMoOÆ2HO,
Correspondence to H Kondo, Department of Biochemical
Engineering and Science, Kyushu Institute of Technology,
Kawazu 680-4, Iizuka 820-8502, Japan.
Fax: + 81 948 7801, Tel.: + 81 948 29 7814,
E-mail: kondo@bse.kyutech.ac.jp
Abbreviations: APAD, acetylpyridine adenine dinucleotide; APADH,
reduced form of APAD; DON, 6-diazo-5-oxo- L -norleucine.
(Received 7 October 2003, revised 2 December 2003,
accepted 23 December 2003)
Trang 2200 lg; KI, 100 lg; H3BO3, 500 lg; biotin, 2 lg; inositol,
10 mg; nicotinic acid, 0.2 mg and calcium pantothenate,
0.2 mg, per litre] Where indicated, the following
supple-ments were added to the synthetic media at a final
concentration of 20 mgÆL)1: histidine, leucine, tryptophan
and pyridoxine Solid media contained 2% agar For
each liquid culture experiment, yeast cells were shaken at
250 r.p.m in 10 mL of medium at 30C for the period of
time specified LB medium [1% polypeptone, 0.5% NaCl
and 0.5% yeast extract (v/v/v)] was used to grow E coli
General procedures
All of the gene manipulations of S cerevisiae and E coli
were carried out using standard methods [13]
Chromo-somal DNA from S cerevisiae was prepared according to
the literature [14] Yeast genomic DNA sequences were
retrieved from a database by using the website (http://
genome-www.stanford.edu/Saccharomyces/) PCR was run
on an Astec PC-700 (Tokyo, Japan) or Biometra T-gradient
thermoblock (Gottingen, Germany) Sequencing of
plas-mids was carried out by the dideoxy chain termination
method on an automatic DNA sequencer model DSQ1000
from Shimadzu (Kyoto, Japan) Protein sequences were
determined on an Applied Biosystems 491 A sequencer
Mutant construction
The pyridoxine auxotrophic mutants of yeast were
pro-duced by ethyl methanesulfonate mutagenesis [15] In brief,
cells were treated with 3% ethyl methanesulfonate for
50 min and then spread over the entire surface of synthetic
medium plates supplemented with pyridoxine and the
growth requirements, and the plates were incubated at
30C The colonies that appeared on the supplemented
plates were then transferred by replica plating first to a
minimal plate without pyridoxine (–PN) and then to one
supplemented with pyridoxine (+PN) The plates were
scored for colonies that appeared on +PN media but not
on the –PN media This selection process was repeated
several times to ensure that the colonies that were unable to
grow in –PN medium are indeed pyridoxine auxotrophs
Transformation of yeast cells
Pyridoxine auxotrophic mutants were transformed with a
YEpM4-based genomic library [11] for complementation
by the lithium acetate protocol [16] Transformants were
selected on synthetic media lacking pyridoxine and leucine
The plasmids were isolated from S cerevisiae as described
[14]
Construction of over-expression plasmids for theSNO1
andSNZ1 genes
The coding regions of the SNO1 (672 bp) and SNZ1
(891 bp) genes were amplified by PCR in one step with the
oligonucleotides shown in Table 1 as primers Both of the
genes were constructed either with or without a His-tag at
the 3¢ terminus of the coding region Thus, the stop codon of
SNO1and SNZ1 was replaced with bases coding for the
sequence LEHHHHH as a C-terminal extension The PCR
was run as follows: After heating at 94C for 5 min, the following cycle was repeated 25 times; 94C, 1 min; 55 C,
1 min; 70C, 1–1.5 min, and finally heated at 72 C for
5 min Each PCR product was purified by agarose gel electrophoresis before ligation into pCR2.1-TOPO (Invi-trogen) After confirming the correct DNA sequence, the coding region of SNO1 and SNZ1 was excised from the plasmid and, where the His-tag was present, recloned into the NdeI/XhoI sites of pET21a or pET24a, respectively Where a His-tag was absent, the coding region of both genes was recloned into the NcoI/BamHI sites of pET21d The resulting recombinant plasmids are termed pSNO1H, pSNZ1H, pSNO1 and pSNZ1, respectively (Table 2) Protein production and purification
The two genes were expressed in E coli BL21(DE3) (Novagen) separately or simultaneously following trans-formation with one or two of the plasmids prepared above Transformants were grown in 1 L of LB medium
in the presence of ampicillin (50 lgÆmL)1), kanamycin (30 lgÆmL)1) or both, to late logarithmic phase, whereupon isopropyl thio-b-D-galactoside was added to 0.4 mM, except for the expression of Sno1p (0.1 mM) Eight hours after induction the cells were collected by centrifugation and washed with phosphate buffered saline Subsequent steps of protein purification were carried out at 4C, unless otherwise stated
The complex of Sno1p with a His-tag and Snz1p without a tag The washed cells were resuspended in 100 mL of
5 m imidazole, 0.5 NaCl, 20 m Tris/HCl, pH 8.0
Table 1 Sequences of oligonucleotides used as PCR primers Symbols
O, Z and H stand for Sno1p, Snz1p and His-tag, respectively Underlined are the restriction enzyme sites.
Primer Sequence P1O 5¢-ATACCATGGACAAAACCCACAGTACAATG P1OH 5¢-CATATGCACAAAACCCACAGTAC
P2O 5¢-TATGGATCCTTAATTAGAAACAAACTGTCTGA
TAAAC P2OH 5¢-CTCGAGATTAGAAACAAACTGTCTGATAAACC P1Z 5¢-ATACCATGGCTGGAGAAGACTTTAAGATC P1ZH 5¢-CATATGACTGGAGACTTTAAGATC P2Z 5¢-TATGGATCCTCACCACCCAATTTCGGAAAG P2ZH 5¢-CTCGAGCCACCCAATTTCGGAAAGT
Table 2 Over-expression plasmids for Sno1p and Snz1p prepared in this study Symbols O, Z and H stand for Sno1p, Snz1p and His-tag, respectively.
Name of plasmid
Primer
Vector Forward Backward
Trang 3(buffer A) containing 5 mM glutamine, 0.4 mM
phenyl-methanesulfonyl fluoride and 0.2 mL of dimethylsulfoxide
The suspension was sonicated and then centrifuged at
17 000 g for 30 min The supernatant containing 120 mg of
protein was filtered through a 0.45 lm filter and applied to
a His-Bind affinity column (1· 10 cm), pretreated with
50 mMNiSO4and equilibrated with buffer A The column
was washed successively with buffer A and buffer B
(50 mMimidazole, 0.5M NaCl, 20 mM Tris/HCl, pH 8.0)
and protein was finally eluted with a gradient of
100–500 mMimidazole in buffer B The protein-containing
fractions were pooled and dialyzed against 35 mM
potas-sium phosphate, 1 mMEDTA and 0.1 mM dithiothreitol,
pH 7.5 The purity of the desired protein was greater than
95% at this stage and the yield was 25 mg from a 1 L
culture Sno1p and Snz1p with a His-tag were purified
analogously with a yield of 30 mg each from 1 L of culture
Protein concentration was determined on the basis of the
molar extinction coefficient of 15.9 and 12.3 mM )1Æcm)1at
280 nm for Sno1p and Snz1p, respectively, deduced from
their amino acid compositions
Sno1p without a His-tag The washed cells were
resus-pended in 50 mL of 10 mMpotassium phosphate buffer and
1 mM EDTA, pH 7.0 The suspension was sonicated and
then centrifuged at 17 000 g for 20 min The pellet was
taken up in 50 mL of the same buffer and 2 mL of Triton
X-100 and shaken at room temperature for 30 min The
suspension was centrifuged (17 000 g) for 30 min and the
supernatant discarded This process was repeated twice
more for the pellet The washed pellet was mixed with
10 mL of 50 mMTris/HCl containing 10 mMdithiothreitol
and 8 M urea, pH 9.0, shaken at 37C for 1 h and
centrifuged for 30 min The supernatant was mixed with
30 mL of the same buffer with 6M urea and dialyzed
against the same buffer with 4M urea Dialysis was
continued against the buffers containing 2, 1 and 0Murea,
successively The solution was concentrated to one half the
volume by dialysis against 50 mMTris/HCl, 1 mM
dithio-threitol and 0.1 mM EDTA, pH 9.0, containing 15%
polyethylene glycol 20 000 and subjected to gel filtration
chromatography on Superdex 200 (2.6· 60 cm,
Pharma-cia) to give 30 mg of virtually pure protein
Snz1p without a His-tag Harvested cells were processed
in a way identical to that for Sno1p up to the cell
disruption step The supernatant was subjected to
DEAE-cellulose chromatography (2· 10 cm, Whatman,
Maid-stone, UK) and protein was eluted with a linear gradient of
0–500 mM NaCl in 50 mMpotassium phosphate, pH 7.0
Snz1p was eluted at about 200 mM salt The pooled
fractions (25 mL) were concentrated and then subjected to
gel filtration chromatography as described above to give
14 mg of the desired protein
Glutaminase assay
The glutaminase activity of the complex of Sno1p with a
His-tag and Snz1p was determined in two steps Thus,
glutamate formed was converted to 2-oxoglutarate by
glutamate dehydrogenase with acetylpyridine adenine
dinu-cleotide (APAD) as cosubstrate [17] The glutaminase
reaction was carried out in 0.3 mL of 50 mM Tris/HCl,
pH 8.0, in the presence of 1–10 mMglutamine and 30 lg of the complex at 30C for 10 min The sample was then boiled for 1 min and kept frozen at)80 C for subsequent analysis To quantitate the amount of glutamate formed, 0.3 mL of the sample was incubated in 1 mL of 50 mMTris/ HCl, pH 8.0, containing 1 mMEDTA, 0.5 mMAPAD and
7 units of glutamate dehydrogenase at 37C for 90 min After centrifugation (14 000 g) for 1 min, the absorbance of the supernatant was read at 363 nm The absorbance of the reduced form of APAD (APADH) was linear over the 2–100 lM range of glutamate with a molar extinction coefficient of 8900M )1Æcm)1[17]
Detection of glutamate Glutamate formed from glutamine by the complex of Sno1p with a His-tag and Snz1p, was further detected by TLC following dansylation The reaction mixture (0.3 mL) contained 10 mM glutamine, 30 lg of the complex in
50 mMTris/HCl, pH 8.0, and it was incubated at 30C for
10 min Ten microliter aliquots of this solution together with 10 lL each of 10 mMglutamine and glutamate were separately allowed to react with dansyl chloride under standard conditions [18] Aliquots (1.5 lL each) were spotted on silica gel 60 F254(Merck, Darmstadt, Germany) and developed in chloroform-t-amyl alcohol-glacial acetic acid (70 : 30 : 3, v/v/v) The dansylated products were visualized under ultraviolet light
Inhibition of Sno1p with DON Inhibition of the complex of Sno1p with a His-tag and Snz1p by 6-diazo-5-oxo-L-norleucine (DON) was studied according to the literature [19] The reaction mixture contained 0.15 mg of the complex and various concentra-tions (1–10 mM) of DON in 1 mL of 50 mM Tris/HCl,
pH 8.0, and was incubated at 30C Aliquots (200 lL) removed at a specified time were mixed with other ingredients for glutaminase assay and the remaining activity
of the complex was determined as described above
Results
Yeast pyridoxine auxotrophs Pyridoxine auxotrophs of S cerevisiae were prepared by ethyl methanesulfonate mutagenesis Several strains were tested including D373-1 and in all the cases auxotrophs showing clear pyridoxine requirements were obtained (data not shown) After several rounds of screening, around 20 mutants were established from D373-1 Among these was mutant K64 (Fig 1) To identify the gene(s) affected by complementation, the mutant was transformed with a library of yeast chromosomal DNA It was found that 4.6 and 5.1 kb overlapping fragments of chromosome XIII carrying SNO1 and SNZ1 were capable of complementing the defect of mutant K64 (Figs 1 and 2) They are homologues of PDX2 and PDX1 of fungus C nicotianae, respectively [7,20], strongly suggesting that they are the genes responsible for the pyridoxine auxotrophy observed for that mutant
Trang 4Site of mutation in K64
In order to identify the site of mutation, the two genes were
amplified by PCR with the chromosomal DNA of mutant
K64 as template Sequencing of the mutated genes revealed
that there was indeed a mutation on both of the genes In
SNO1, the 199th G from the 5¢ terminus of the open reading
frame was deleted to result in a frame-shift and appearance
of stop codons in the downstream As a result, a protein as
small as 70 residues is generated, a size too small for any
protein to be functional as an enzyme (see below) In SNZ1,
the 709th G was converted to A to result in replacement of
Gly237 with Arg It is noted that this residue and the
surrounding regions are well conserved among the
homo-logues of SNZ1 including pyroA and PDX1 [6,7], suggesting
that the residue plays an important role It should be
emphasized that the dual mutation of the two genes was
necessary to make yeast cells pyridoxine auxotrophic; cells
were still viable in the absence of pyridoxine even when
either one of the genes was disrupted separately (data not
shown) This observation is consistent with those reported
previously [21] Presumably, their homologues SNO2,
SNO3, SNZ2 and SNZ3 complement the defect The
relationship of all of these genes remains to be clarified
Expression and purification of Sno1p and Snz1p
In light of the similarity in the amino acid sequence of
Sno1p to that of the glutaminase subunit of imidazole
glycerol phosphate synthase [22], Sno1p may possess the
ability to hydrolyze glutamine In addition, in reference to
the observation that Sno1p and Snz1p act together in the stationary phase of yeast cells [8] and the result of two-hybrid analysis [23], they may form a complex To assess these possibilities, we embarked on a characterization of the two proteins
Over-expression plasmids were prepared for both genes with or without a C-terminal His-tag (Table 2), as detailed under Experimental procedures E coli cells were trans-formed with one or two of the recombinant plasmids The resulting cells over-expressed the desired protein(s) up to 15% of the total cellular proteins When Sno1p was expressed alone, it was kept in the inclusion body, irrespective of the occurrence of a His-tag (data not shown) Whereas, Snz1p, expressed either alone or coexpressed with Sno1p, was found in the soluble fractions Although purification procedures differed slightly from sample to sample, depending on the occurrence of a tag and the location of the protein in question, Sno1p, Snz1p and their complex were eventually purified to near homogeneity (Fig 3) Typically, 30, 30 and 25 mg of protein were
Fig 1 Growth of S cerevisiae strains D373-1, K64 and K64t Yeast
cells were treated with ethyl methanesulfonate as detailed under
Experimental procedures After several rounds of screening on media
containing pyridoxine (+PN) and not containing pyridoxine (–PN),
auxotrophic mutants were established, one of which was K64 K64t
represents the transformant harboring plasmid pDYX11 (Fig 2) and
is capable of growing in –PN medium.
Fig 2 Partial map of S cerevisiae chromosome XIII carrying SNO1 and SNZ1 Dark grey bars represent 4.6 and 5.1 kb DNA fragments capable of complementing the pyridoxine auxotrophy of mutant K64.
Fig 3 SDS/PAGE of purified Sno1p, Snz1p and their complex with or without a C-terminal His-tag Lane 1, protein markers; lane 2, Sno1p without tag; lane 3, Sno1p with tag; lane 4, complex of Sno1p with tag and Snz1p without tag; lane 5, Snz1p without tag; lane 6, Snz1p with tag About 10–20 lg of protein was loaded and stained with Coo-massie Brilliant blue.
Trang 5obtained from a 1 L culture for Sno1p, Snz1p and the
complex, respectively Identity of each protein was
con-firmed by N-terminal sequencing; the sequences were
correct at least to the 9th cycle from the N-terminus
including the initiating Met: MHKTHSTMS for Sno1p and
MTGEDFKIKS for Snz1p
Properties of the complex of Sno1p and Snz1p
When Sno1p with a His-tag and Snz1p without a tag were
coexpressed and then applied to a His-Bind affinity column,
not only Sno1p but also Snz1p bound to the column and
was eluted simultaneously by imidazole, strongly suggesting
that they form a complex The ratio of the two proteins,
assessed by SDS/PAGE, seemed to be equimolar, although
a more rigorous assessment is necessary to prove this
assertion The glutaminase activity of this complex was
assessed in two ways First, the glutamate formed was
oxidized to 2-oxoglutarate by APAD and glutamate
dehydrogenase, and the generated APADH was determined
spectroscopically Second, the glutamate formed was
dansy-lated and analyzed by TLC (Fig 4) These experiments
consistently pointed to the formation of glutamate, proving
unambiguously that the complex did indeed hydrolyze
glutamine efficiently
Kinetics of the glutaminase reaction
The kinetics of glutamine hydrolysis mediated by the
complex of Sno1p with a His-tag and Snz1p was determined
at 30C The reaction rate was proportional to protein
concentration over a 5–50 lg range and it was constant up
to at least 1 h (data not shown) The reaction followed the
simple Michaelis–Menten formulation as illustrated in
Fig 5 The specific activity (Vmax) and Kmfor glutamine
obtained from analysis of these data were 0.48 lmolÆ
min)1Æmg)1 and 3.4 mM, respectively These values seem
to be reasonable for a glutaminase, though the specific
activity varies drastically from enzyme to enzyme and
depends on whether a proper synthetase partner and cosubstrate are present or not For example, the Vmaxand
Km values of imidazole glycerol phosphate synthase in the absence of substrate are 0.084 lmolÆmin)1Æmg)1 and 4.8mM, respectively [22] The Vmaxwas enhanced 39-fold and Kmlowered 20-fold in the presence of substrate N1 -[(5¢-phosphoribulosyl)formimino]-5-aminoimidazole 4-carbox-amide ribonucleotide It may be reasonable therefore to assume that once the unknown substrate or ligand for Snz1p was added, the glutaminase activity could have been even higher It should be noted that the enzyme activity is gradually lost over time with a half life of 2–3 days at 4C Several additives such as ATP were tested as a stabilizer, but thus far we have not been successful in stabilizing the enzyme activity Incidentally, no glutaminase activity was found for Snz1p alone
The function of Sno1p The data shown above revealed that the complex of Sno1p and Snz1p is a glutamine amidotransferase, with the former serving as the glutamine-hydrolyzing machinery This latter notion is based solely on the sequence homology of Sno1p with the glutaminase subunit of imidazole glycerol phos-phate synthase To test this hypothesis directly, Sno1p and Snz1p were separately expressed, purified and characterized
as detailed under Experimental procedures As described above, Sno1p expressed in E coli resided in the inclusion body and hence use of Triton X-100 and urea was necessary for its solubilization Even after renaturation, however, Sno1p exhibited no glutaminase activity, nor did the addition of 0.5 to 3-fold Snz1p have any effect It was only when Sno1p and Snz1p were partially denatured together and then renatured, that glutaminase activity was observed Thus, the two proteins were mixed in 8 urea in various
Fig 4 TLC analysis of glutamate formed from glutamine mediated by
the complex of Sno1p with a His-tag and Snz1p Glutamate, glutamine
and the reaction mixture were dansylated separately and aliquots
(2.5 nmol each) were applied on silica gel 60 F 254 and developed in
chloroform-t-amyl alcohol-glacial acetic acid (70 : 30 : 3, v/v/v) The
dansylated (DANS) products were visualized under ultraviolet light.
Fig 5 Michaelis–Menten kinetics for glutamine hydrolysis mediated by the complex of Sno1p with a His-tag and Snz1p The reaction was carried out in 1 mL of 50 m M Tris/HCl, pH 8.0, in the presence of 1–10 m M glutamine and 50 lg of the complex, at 30 C for 10 min The sample was then boiled for 1 min and a 0.3 mL aliquot was incubated in 1 mL of 50 m M Tris/HCl, pH 8.0, containing 1 m M
EDTA, 0.5 m M APAD and 7 units of glutamate dehydrogenase at
37 C for 90 min After centrifugation for 1 min, the absorbance of the supernatant was read at 363 nm for APADH The curve drawn is a theoretical one based on 0.48 lmolÆmin)1Æmg)1for V max and 3.4 m M
for K m for glutamine.
Trang 6molar ratios (0.2 : 4 of Sno1p : Snz1p) The urea
concen-tration was lowered gradually to 6, 4, 2, and 1Mby dialysis
Finally, the enzyme solution was dialyzed against buffer
without urea, and glutaminase activity was determined The
mixtures of Sno1p and Snz1p with a molar ratio of the
former greater than 0.5 exhibited partial activity; at a
maximum of 5% of that of the intact complex described
above This suggests that although Sno1p and Snz1p
associate spontaneously, renaturation of either one or both
of the proteins was incomplete and the complex formation is
slow and/or incomplete under the conditions employed
Inhibition of the glutaminase activity by DON
Glutamine amidotransferases are inhibited irreversibly by
6-diazo-5-oxo-L-norleucine (DON) [24] Hence, the
suscep-tibility of the complex of Sno1p and Snz1p to DON was
studied under conditions detailed under Experimental
procedures As shown in Fig 6, DON deactivated the
enzyme in a time- and dose-dependent manner Typically, the half-life of deactivation was 10 min at 10 mMDON at
30C It is noted that the inhibition did not go to completion even at the highest concentration of DON employed but halted at a point where about half of the enzyme activity is lost The reason for this phenomenon is not known but a similar observation was made for phosphoribosylpyrophos-phate amidotransferase [24] Glutamine was effective in protecting the enzyme from inactivation and its effect was again dose-dependent, suggesting that inhibition by DON occurs at the active site or the glutamine-binding site of the enzyme (Sno1p) Although DON inhibition of Sno1p was not pursued further, it is worth pointing out that the cysteine serving as the key catalytic residue and modified covalently by DON in other amidotransferases is also conserved in Sno1p at position 100
Discussion
As described above, the gene products of SNO1 and SNZ1 serve as a glutamine amidotransferase, which is needed
to supply ammonia as a source of the ring nitrogen of pyridoxine [4] Although Sno1p alone does not exhibit detectable glutaminase activity, it seems certain that it is responsible for the hydrolysis of glutamine mediated by the complex with Snz1p For example, the amino acid sequence
of Sno1p has 40% identity to that of the glutaminase subunit of yeast imidazole glycerol phosphate synthase [22]
In addition, the key catalytic residues required for glutamine hydrolysis by glutamine amidotransferases including imi-dazole glycerol phosphate synthase, i.e Cys100, His203 and Glu205 (numbering based on Sno1p), are conserved in Sno1p as well Presumably, Sno1p hydrolyzes glutamine by the same mechanism as those of other glutamine amido-transferases such as imidazole glycerol phosphate synthase and carbamoyl-phosphate synthetase, whose three-dimen-sional structures are available [25,26]
In light of the function of these glutamine amidotrans-ferases, Snz1p may be a synthetase that is mediating the coupling of ammonia released from glutamine with an unknown acceptor substrate In reactions of many amido-transferases, ammonia is delivered from the site of forma-tion to the site of coupling through a tunnel that is present
in the synthetase subunit [25,26] It is hence tempting to assume that there is such a tunnel in Snz1p as well, although this notion awaits experimental verification of the structure
Fig 6 Inhibition of the glutaminase activity of the complex of Sno1p
with a His-tag and Snz1p by 6-diazo-5-oxo- L -norleucine (DON) The
reaction mixture contained 150 lg of the complex and no (s), 1 (e), 5
(h,) and 10 m M (n) DON in the absence (open symbols) and presence
(j) of 10 m M glutamine in 1 mL of 50 m M Tris/HCl, pH 8.0, at 30 C.
Aliquots (200 lL) were withdrawn at specified times and the remaining
activity of the complex was determined.
Scheme 1 Putative synthetic pathway to pyridoxine in yeast Ammonia released from glutamine by Sno1p condenses with an unknown acceptor (?)
or a derivative of ribulose 5-phosphate This process is mediated by Snz1p but the subsequent steps remain obscure.
Trang 7by means of X-ray crystallography Incidentally, the amino
acid sequence of Snz1p does not show homology to any
other known proteins and hence its three-dimensional
structure could be unique In addition, once the
amido-acceptor substrate of Snz1p is identified, its addition to the
reaction system will enhance the glutaminase activity of
Sno1p significantly In light of the fact that a ketopentose
seems to be a component of the skeleton of pyridoxine in
yeast and related organisms (see below), dihydroxyacetone
phosphate, glyceraldehyde 3-phosphate or related
com-pounds are probable candidates for the coupling partner
These possibilities are presently under scrutiny in this
laboratory
Recently, it was shown that a ketopentose is one of the
starting materials for pyridoxine in yeast and the initial form
of vitamin B6 produced is 2¢-hydroxypyridoxine [9,10] Our
unpublished observation supports these findings; the gene
RKI1 coding for ribose 5-phosphate ketol-isomerase
(Rki1p), which interconverts ribose 5-phosphate and
ribu-lose 5-phosphate, dictates somehow pyridoxine synthesis in
yeast In this light, ribulose 5-phosphate may be the more
probable candidate for the starting material, as its structure
fits the skeleton from positions 2¢ to 4¢ of
2¢-hydroxypyri-doxine neatly Hence, the possibility that ribulose
5-phos-phate serves as the direct ammonia-acceptor was addressed
It was found that this compound considerably inhibits the
glutaminase activity of the complex of Sno1p and Snz1p in a
competitive fashion; the activity decreased to 70% at 8mM
Ribose 5-phosphate was as equally effective as ribulose
5-phosphate but dihydroxyacetone phosphate was without
effect These data seem to suggest that, although it does
interact with the glutaminase complex, ribulose 5-phosphate
is not the direct acceptor of ammonia
Based on these arguments, the following scheme is
proposed for the synthetic route to pyridoxine in yeast and
related organisms (Scheme 1) Ammonia released from
glutamine undergoes condensation with either a derivative
of ribulose 5-phosphate or an unknown acceptor with a
three-carbon unit to give an aminated intermediate These
two components eventually undergo coupling and
cycliza-tion to form 2¢-hydroxypyridoxine If this scheme holds, the
synthetic pathways to pyridoxine of yeast and related
organisms are remarkably similar to that of E coli, as both
utilize a ketopentose or its derivative as the starting material
Although it seems certain that
3-amino-1-phosphohydroxy-propan-2-one, a key component in the E coli pathway and
generated by the product of gene pdxA, is not involved,
compounds related to it structurally could be the coupling
partner of the ketopentose in yeast
One characteristic feature of the genes SNO1 and SNZ1
is that they act simultaneously in the stationary phase of
yeast cells [8] They are assumed to participate in the
metabolism of nucleotides In this context, the assertion that
pyridoxine may be the precursor of the pyrimidine moiety of
thiamin in yeast is intriguing [27] In fungi, pyroA and SOR1
(PDX1), homologues of yeast SNZ1, play a defensive role
against reactive oxygen species such as singlet oxygen [6,7]
Taken together, pyridoxine synthesis in various organisms
ranging from Bacillus to plants seems to play a broader
physiological role than simply supplying the cofactor that is
essential for amino acid metabolism
Acknowledgements
The expert technical assistance of Ms Miwa Kitamura is gratefully acknowledged This work was supported in part by a grant from the Regional Science Promotion Program of Japan Science and Technol-ogy Corporation.
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