tuberculosis, the etiologic agent of tuberculosis, encodes a protein Rv1771 that shows 32% identity with the rat l-gulono-1,4-lactone oxidase protein.. tuberculosis produces a novel, hig
Trang 1enzyme for the synthesis of vitamin C,
Beata A Wolucka1and David Communi2
1 Laboratory of Mycobacterial Biochemistry, Pasteur Institute of Brussels, Institute of Public Health, Belgium
2 Institute of Interdisciplinary Research, IRIBHM, Faculty of Medicine, Free University of Brussels, Belgium
Vitamin C (l-ascorbic acid; L-AA) is an important
metabolite of plants and animals It functions as an
antioxidant (or pro-oxidant), an enzyme cofactor, an
effector of gene expression, and a modulator of
react-ive oxygen species (ROS)-mediated cell signaling
L-AA is therefore involved in a wide array of crucial
physiologic processes, including: biosynthesis of
colla-gen and other hydroxyproline⁄ hydroxylysine-containing
proteins⁄ peptides; synthesis of secondary metabolites,
hormones and cytokines [1]; oxidative protein folding
and endoplasmic reticulum stress [2]; cell proliferation
and apoptosis [3]; activation of the epithelial cystic
fibrosis transmembrane conductance regulator chloride channel [4] and of surfactant production in human lungs [5]; macrophage function [6]; immune homeosta-sis [5]; and stress rehomeosta-sistance
Plants synthesize ascorbic acid via de novo and sal-vage pathways [7], whereas a de novo pathway invol-ving UDP-d-glucuronic acid operates in animals [8]
l-Gulono-1,4-lactone is a direct precursor of vitamin C
in animals [8], but also in plants [9] and in some pro-tists [10] In plants, L-AA can be formed additionally from l-galactono-1,4-lactone by a highly specific mito-chondrial dehydrogenase (EC 1.3.2.3) [11,12] The
Keywords
ascorbic acid; biosynthesis; L -gulonolactone
oxidase; tuberculosis; vitamin C
Correspondence
B A Wolucka, Laboratory of Mycobacterial
Biochemistry, Pasteur Institute of Brussels,
642 Engeland Street, B-1180 Brussels,
Belgium
Fax: +32 2 373 3282
Tel: +32 2 373 3100
E-mail: bwolucka@pasteur.be
(Received 21 June 2006, accepted 31 July
2006)
doi:10.1111/j.1742-4658.2006.05443.x
The last step of the biosynthesis of l-ascorbic acid (vitamin C) in plants and animals is catalyzed by l-gulono-1,4-lactone oxidoreductases, which use both l-gulono-1,4-lactone and l-galactono-1,4-lactone as substrates l-Gul-ono-1,4-lactone oxidase is missing in scurvy-prone, vitamin C-deficient ani-mals, such as humans and guinea pigs, which are also highly susceptible to tuberculosis A blast search using the rat l-gulono-1,4-lactone oxidase sequence revealed the presence of closely related orthologs in a limited num-ber of bacterial species, including several pathogens of human lungs, such
as Mycobacterium tuberculosis, Pseudomonas aeruginosa, Burkholderia cepa-cia and Bacillus anthracis The genome of M tuberculosis, the etiologic agent of tuberculosis, encodes a protein (Rv1771) that shows 32% identity with the rat l-gulono-1,4-lactone oxidase protein The Rv1771 gene was cloned and expressed in Escherichia coli, and the corresponding protein was affinity-purified and characterized The FAD-binding motif-containing Rv1771 protein is a metalloenzyme that oxidizes l-gulono-1,4-lactone (Km5.5 mm) but not l-galactono-1,4-lactone The enzyme has a dehydroge-nase activity and can use both cytochrome c (Km4.7 lm) and phenazine methosulfate as exogenous electron acceptors Molecular oxygen does not serve as a substrate for the Rv1771 protein Dehydrogenase activity was measured in cellular extracts of a Mycobacterium bovis BCG strain In con-clusion, M tuberculosis produces a novel, highly specific l-gulono-1,4-lac-tone dehydrogenase (Rv1771) and has the capacity to synthesize vitamin C
Abbreviations
GST, glutathione-S-transferase; IPTG, isopropyl thio-b- D -galactoside; L-AA, L -ascorbic acid; MALDI Q-TOF, MALDI quadrupole TOF;
ROS, reactive oxygen species.
Trang 2oxidation of l-gulono-1,4-lactone to L-AA in animals
is catalyzed by an oxygen-dependent enzyme,
l-gul-ono-1,4-lactone oxidase (EC 1.1.3.8) [13] In plants [9]
and in Euglena [10], the oxidation involves ill-defined
l-gulono-1,4-lactone dehydrogenases that use
cyto-chrome c and phenazine methosulfate respectively, as a
direct electron acceptor The animal and plant
l-gul-onolactone oxidoreductases are also active towards the
l-galactono-1,4-lactone substrate
Only scarce data are available on the presence of
ascorbic acid in lower eukaryotes Fungi do not contain
L-AA but rather its 5-carbon homolog,
d-erythroascor-bic acid [14] Two apparently different
l-gulono-1,4-lac-tone oxidase activities were detected in yeasts One of
the enzymes oxidizes l-galactono-1,4-lactone but not
l-gulono-1,4-lactone [15] The other enzyme (ALO1)
has a broader specificity and uses
d-arabinono-1,4-lac-tone [16], l-galactono-1,4-lacd-arabinono-1,4-lac-tone and
l-gulono-1,4-lac-tone [17] as substrates d-Arabinono-1,4-lacl-gulono-1,4-lac-tone is a
natural substrate in the pathway to d-erythroascorbic
acid [14,18] Like yeasts, a protozoan parasite
Try-panosoma brucei possesses a d-arabinono-1,4-lactone
oxidase that can also oxidize l-galactono-1,4-lactone
but not l-gulono-1,4-lactone [19]
The sequence of the gene for rat
l-gulono-1,4-lac-tone oxidase (GLO) is known [20] Several genes for
putative l-gulono-1,4-lactone dehydrogenase
isoen-zymes have been identified in plants [9] The gene
encoding the d-arabinono-1,4-lactone oxidase (ALO1)
of yeasts [18] shows significant homology with the
genes for both the rat l-gulono-1,4-lactone oxidase
and the plant mitochondrial l-galactono-1,4-lactone
dehydrogenase [12,20] Humans and some animals
(including other primates and guinea pigs) are natural
mutants for ascorbic acid synthesis because of the
non-functional GLO gene for l-gulono-1,4-lactone oxidase
[8] Consequently, they require vitamin C in the diet to
prevent scurvy
Little is known about the presence of vitamin C and
related biosynthetic enzymes in bacteria Exogenous
l-gulono-1,4-lactone can be converted to L-AA by
unspecific, heteromeric dehydrogenases of
Gluconobact-er oxydans and of Acetobacter suboxydans [21], which
are also active towards d-xylose and some hexoses
Although interesting from a biotechnological point of
view, these enzymes are not related to the known
l-gulono-1,4-lactone oxidase proteins and their
physio-logic role is unknown
Surprisingly, the genome of Mycobacterium
tubercu-losis, the causative agent of tubercutubercu-losis, encodes a
protein (Rv1771) that is similar to the rat
l-gulono-1,4-lactone oxidase In the present work, we cloned
and expressed the Rv1771 gene, and showed that it
encodes a novel l-gulono-1,4-lactone dehydrogenase of the M tuberculosis complex
Results Heterologous expression and purification of the recombinantL-gulono-1,4-lactone dehydrogenase (Rv1771) of M tuberculosis
The Rv1771 DNA was cloned into the pDEST15 vec-tor by using the Gateway system, and the obtained pDEST15_Rv1771 plasmid was used for expression
of the recombinant glutathione-S-transferase (GST) fusion protein in Escherichia coli The recombinant protein contained an engineered enterokinase cleavage site in the junction between the GST tag and the Rv1771 sequence Upon 3 h of induction of the pDEST15_Rv1771 E coli strain with isopropyl-b-d-thiogalactopyranoside (IPTG) at 37C, the yield of the recombinant Rv1771 protein was very low (0.1 mg per liter of culture) Longer inductions (16 h) at a lower temperature (26C) resulted in complete loss of the recombinant protein, probably due to proteolytic degradation (results not shown) Similarly, omission of Triton X-100 from the extraction buffer resulted in lower yields of the recombinant Rv1771 protein, thus suggesting that the detergent helps solubilize the pro-tein The affinity-purified protein was concentrated by using Strataclean resin, as described in Experimental procedures SDS⁄ PAGE revealed the presence of one protein band of about 70 kDa (Fig 1, lane 2) The identity of the protein was confirmed by enterokinase treatment After digestion of the affinity-purified GST-tagged protein with enterokinase followed by concen-tration, the 70 kDa band disappeared, and two new bands of 45 and 26 kDa were seen on SDS⁄ PAGE that corresponded to the mycobacterial Rv1771 pro-tein and the freed GST tag, respectively (Fig 1, lane 3) The observed molecular masses for the GST-tagged and free forms of the l-gulono-1,4-lactone dehydroge-nase were slightly lower than those expected (74 and
48 kDa, respectively), presumably because of either limited proteolysis or abnormal migration In contrast, shortening the induction time to 1 h resulted in about
10 times higher yields of the recombinant Rv1771 pro-tein (Fig 1, lane 4) However, under these conditions, the GST-affinity eluate contained the recombinant GST fusion protein (the identified tryptic peptides are ALGPQLAQR, LGLENQGDVDPQSITGATATATH GTGVR, FQNLSAR, SDEQPKPTPGWQR, FTEM
YGR), which was accompanied by a host-derived Hsp60 chaperone protein (the identified tryptic peptide
Trang 3was AAVEEGVVAGGGVALIR), as determined by
combined MALDI-TOF MS of trypsin-digested
pro-tein bands (Fig 1, lane 4; Fig 2) and western analysis
with anti-GST IgG (Fig 1, lane 5) Copurification of
the mycobacterial l-gulono-1,4-lactone dehydrogenase
with the Hsp60 heat-shock protein might reflect
physi-ologic protein–protein interactions, as proposed for the
plant Hsc70.3 cognate heat-shock protein and another
vitamin C-related enzyme, the
GDP-mannose-3¢,5¢-epimerase [9] The presence of multiple
GST-contain-ing bands of about 30 kDa (Fig 1, lane 5) suggests
that an important portion of the fusion protein was
degraded by the host proteases On the other hand,
attempts to produce a His-tagged version of the
Rv1771 protein by using the pRSETc or the Gateway pDEST17 expression vectors were unsuccessful
Characterization of the recombinant
L-gulono-1,4-lactone dehydrogenase of
M tuberculosis The Rv1771 gene of M tuberculosis encodes a 428 amino acid protein (Fig 2) that shows 32% identity with the rat l-gulono-1,4-lactone oxidase and 22–24% identity with the putative plant l-gulono-1,4-lactone dehydrogenases At2g46740, At2g46750, At2g46760, At5g56490, At5g11540, and At1g32300 [9] The Myco-bacterium bovis genome contains a sequence (Mb1800) identical to the M tuberculosis Rv1771 gene (http:// genolist.pasteur.fr/) A close ortholog of the Rv1771 protein (72% identity) exists in Mycobacterium mari-num (http://www.sanger.ac.uk) In Mycobacterium leprae, a possible pseudogene similar to the
M tuberculosis Rv1771 sequence is present Other my-cobacteria apparently do not contain sequences homol-ogous to the Rv1771 protein The predicted molecular mass of the Rv1771 protein is 48 045 kDa, and the pI
is 7.14 Like the animal and plant l-gulono-1,4-lactone oxidases⁄ dehydrogenases, the M tuberculosis Rv1771 protein possesses in its N-terminus an FAD-binding site (VGSGH49S) with a conserved histidine residue that in the rat l-gulono-1,4-lactone oxidase enzyme (VGGGH54S) participates in the covalent binding of the FAD molecule [22] (Fig 2) Analysis of the dena-tured Rv1771 protein by SDS⁄ PAGE according to the method of Nishikimi et al [23] did not reveal the pres-ence of any fluorescent protein band, thus pointing to the absence of a covalently bound flavin moiety in the recombinant product Moreover, as in the case of the plant l-galactono-1,4-lactone dehydrogenase [12], the native recombinant dehydrogenase of M tuberculo-sis did not show a typical flavin protein absorption spectrum, and addition of exogenous FAD (100 lm)
or riboflavin (1 mm) had no effect on the enzyme
Fig 2 Sequence analysis of the Rv1771
L -gulono-1,4-lactone dehydrogenase of
M tuberculosis The amino acids (16–168)
that form the FAD-binding domain (pfam
designation PF01565) are highlighted in
black The D -arabinono-1,4-lactone oxidase
domain (pfam designation PF04030) (amino
acids 172–427) is highlighted in gray The
position of a potential transmembrane helice
(amino acids 205–227) is indicated by bold
italics Tryptic peptides identified by MALDI
Q-TOF MS are underlined.
Fig 1 Heterologous expression and purification of the recombinant
L -gulono-1,4-lactone dehydrogenase (Rv1771) of M tuberculosis.
SDS ⁄ PAGE of the affinity-purified GST-tagged dehydrogenase
(con-taining an engineered enterokinase cleavage site) obtained from
the E coli host after long (3 h) (lanes 2 and 3) and short (1 h) (lanes
4 and 5) periods of induction with IPTG Fractions obtained with a
long period of induction before (lane 2) and after (lane 3)
enterokin-ase treatment were concentrated on Strataclean beads, as
des-cribed in Experimental procedures Proteins were visualized by
Coomassie blue staining (lanes 1–4) and by western analysis (lane
5) using anti-GST IgG Protein bands (lane 4) were identified by
MALDI-TOF MS of tryptic in-gel digests Lane 1, molecular mass
standards.
Trang 4activity (results not shown) These results suggest
that the mycobacterial dehydrogenase, like the
cauli-flower l-galactono-1,4-lactone dehydrogenase [12], is
not a flavoenzyme The C-terminus of the Rv1771
protein contains a d-arabinono-1,4-lactone domain
(Pfam04030) (Fig 2) that is present in all known
ald-onolactone oxidoreductases
In order to determine the enzymatic activity of the
GST-tagged Rv1771 protein of M tuberculosis, the
oxidase activity was tested in the presence of the
l-gul-ono-1,4-lactone and l-galactl-gul-ono-1,4-lactone substrates,
as described [24], but no activity could be detected
However, the enzyme could oxidize
l-gulono-1,4-lac-tone aerobically by using exogenous cytochrome c from
horse heart as an electron acceptor (Table 1)
l-Galac-tono-1,4-lactone and other sugar derivatives did not
serve as substrates in the dehydrogenase reaction
(Table 1) Interestingly, phenazine methosulfate could
substitute for cytochrome c and was about three times
more efficient as a direct electron acceptor than the
latter (Table 1) 2,6-dichloroindophenol alone could
not serve as electron acceptor in the dehydrogenase
reaction (not shown) Thus, like the plant
l-gulono-1,4-lactone and l-galactono-1,4-l-gulono-1,4-lactone dehydrogenases
[9,12], the mycobacterial enzyme acts exclusively as a
dehydrogenase and does not use molecular oxygen as
an electron acceptor In contrast to the animal and
plant l-gulono-1,4-lactone oxidoreductases, the
myco-bacterial enzyme is specific for l-gulono-1,4-lactone
and has no activity towards l-galactono-1,4-lactone
(Table 1) The steady-state parameters of the
recombin-ant l-gulono-1,4-lactone dehydrogenase of M
tuber-culosis were determined The dehydrogenase obeys
Michaelis–Menten kinetics with l-gulono-1,4-lactone
and cytochrome c as substrates (Fig 3A,B) The
appar-ent Kmvalues for l-gulono-1,4-lactone and cytochrome c
were determined to be 5.5 mm (Fig 3A) and 4.7 lm (Fig 3B), respectively The Vmax value was determined
to be 2.44 lmolÆh)1Æmg protein)1(Fig 3A) The kinetic parameters of the recombinant GST-tagged mycobacte-rial l-gulono-1,4-lactone dehydrogenase are therefore similar to those reported for the plant l-galactonolac-tone dehydrogenase (Km values equal 3.3 mm and 3.6 lm for l-galactono-1,4-lactone and cytochrome c, respectively) [12,25] These results suggest that the mycobacterial enzyme could operate efficiently in vivo Optimal conditions for the mycobacterial dehydroge-nase activity were determined The optimal pH for the dehydrogenase reaction is between 7.5 and 8 (Fig 4A)
At higher pH values, enzyme activity rapidly decreased, probably because of hydrolysis of the lac-tone substrate As for the mammalian l-gulono-1,4-lactone oxidases [26], the temperature optimum for the dehydrogenase reaction was relatively high (39 C) (Fig 4B) Preincubation at 60C for 5 min resulted in only partial inactivation of the enzyme (53% of con-trol), thus indicating that the dehydrogenase is relat-ively heat-stable
The enzyme was completely inhibited by 1 mm N-ethylmaleimide, Cu2+and Zn2+(results not shown) These effects suggest the involvement of sulfhydryl group(s) in the catalytic activity of the mycobacterial enzyme, as observed for the plant l-galactono-1,4-lac-tone dehydrogenase [12,25] No dehydrogenase activity could be measured in the presence of 1 mm potassium cyanide Mg2+ and Ca2+ had no effect on the dehy-drogenase activity, and 1 mm Mn2+ slightly inhibited the enzyme (21% inhibition) However, the mycobacte-rial dehydrogenase requires for its activity trace amounts of a divalent metal ion, because the enzyme was inactive in the presence of 1 mm EDTA
Presence ofL-gulono-1,4-lactone dehydrogenase activity in M bovis BCG strain Copenhagen Crude extracts of exponentially growing M bovis BCG were prepared as described in Experimental proce-dures The dehydrogenase activity could be measured
in the soluble extracts [0.17 mUÆmg protein)1], but not
in the insoluble fraction, because of interfering con-taminants The determined activity of the
mycobacteri-al enzyme was comparable with that reported for crude preparations of plant l-galactono-1,4-lactone dehydrogenase [12,27], Thus, in agreement with previ-ous results [28,29], the Rv1771 protein is expressed in the M tuberculosis complex; it is probably loosely associated with the cell membrane [29], and is enzy-matically active In spite of the presence of dehydroge-nase activity, ascorbic acid could not be detected in
Table 1 Substrate specificity of the recombinant GST-tagged L
-gul-ono-1,4-lactone dehydrogenase of M tuberculosis ND, not
deter-mined All measurements were made in triplicate The limit of
detection was 0.3 mUÆmg protein)1 Mean values ± SD are given.
Substrate
(50 m M )
Enzyme specific activity with different electron acceptors (mUÆmg protein)1)
Cytochrome c (121 l M )
Phenazine methosulfate (2.5 m M )
L -Gulono-1,4-lactone 66.7 ± 4.0 249 ± 17.4
a Measured values were equal to or below the detection limit.
Trang 5the acid extracts obtained from M bovis BCG
Copen-hagen or M tuberculosis H37Rv cells (results not
shown) Possible explanations are that the levels of
extracted ascorbic acid were below the detection limit
of the HPLC method, or that M tuberculosis cells did
not synthesize vitamin C in the in vitro culture
condi-tions used in this study
Discussion
In the present work, we identified a novel l-gulono-1,4-lactone dehydrogenase (Rv1771) of M tuberculosis that catalyzes the reaction depicted in Fig 5 The Rv1771 gene was difficult to express in E coli, and only small quantities of the corresponding GST-tagged protein could be obtained (Fig 1) The enzyme has an absolute specificity for the l-gulono-1,4-lactone sub-strate (Km5.5 mm) (Fig 3A) and shows no activity with l-galactono-1,4-lactone (Table 1) Thus, the mycobacterial enzyme differs from the known l-gul-ono-1,4-lactone oxidases (EC 1.1.3.8), which oxidize both l-gulono-lactone and l-galactono-1,4-lactone [13,17], and also from plant [12], yeast [15] and trypan-osomal [19] l-galactono-1,4-lactone oxidoreductases, which are inactive towards l-gulono-1,4-lactone Because l-galactono-1,4-lactone is not a substrate for the mycobacterial dehydrogenase, we presume that
d-arabinono-1,4-lactone, a five-carbon homolog of
l-galactono-1,4-lactone, is not a substrate either Thus, the mycobacterial dehydrogenase is unusual in its selectivity for l-gulono-1,4-lactone Our preparations
of the recombinant dehydrogenase of M tuberculosis
y = 0,1371x + 28,762
0
5
10
15
20
25
30
35
40
45
-300 -250 -200 -150 -100 -50 0 50 100 150
1/[cyt c] (µ M ) -1
B
y = 135,11x + 24,829
0
10
20
30
40
50
60
-0,3 -0,2 -0,1 0 0,1 0,2 0,3
1/[ L -gulono-1,4-lactone] (m M -1 )
1/V 0
1/V 0
A
Fig 3 Characterization of the recombinant GST-tagged L -gulono-1,4-lactone dehydrogenase (Rv1771) of M tuberculosis Steady-state param-eters of the mycobacterial dehydrogenase determined for: (A) the L -gulono-1,4-lactone substrate in the presence of 121 l M cytochrome c; and (B) the cytochrome c substrate in the presence of 50 m M L -gulono-1,4-lactone Double-reciprocal Lineweaver–Burke plots are shown V0
is lmol of L -gulono-1,4-lactone oxidized per min and per mg of the recombinant dehydrogenase (UÆmg protein)1) L -Gulono-1,4-lactone con-centrations ranged from 5 to 25 m M , whereas cytochrome c concentrations ranged from 24 to 145 l M (B) All measurements were made in duplicate in three independent experiments; the values obtained in a representative experiment are shown.
0
0.02
0.04
0.06
0.08
0.1
pH
Enzyme activity (% of contr
A
B
0
100
200
300
400
500
Temperature (°C)
Fig 4 Effects of pH (A) and temperature (B) on the activity of the
recombinant GST-tagged L -gulono-1,4-lactone dehydrogenase of
M tuberculosis Measurements were made in duplicate; mean
values ± SD are shown In (A), the dehydrogenase activity is
expressed as DA550per min.
O
CH 2 OH
O
H
H O H
O
O H
L -gulono-1,4-lactone
O
CH 2 OH
O
H
H O H
O H O H
L -ascorbic acid
2 cyt c ox 2 cyt c red
Fig 5 Reaction catalyzed by the L -gulono-1,4-lactone dehydroge-nase of M tuberculosis.
Trang 6had low specific activity, ranging from 40 to
66 mUÆmg protein)1 under the nonoptimal
tempera-ture conditions of the enzyme assay (24C) However,
taking into account that the enzyme activity is about
three-fold higher at 39C (Fig 4B) and that the
GST-tagged Rv1771 protein represented only a portion of
the GST affinity-purified fraction (Fig 1, lane 4), the
specific activity of the recombinant dehydrogenase
could be at least 10-fold higher [400–660 mUÆmg
pro-tein)1] The relatively low activity of the recombinant
M tuberculosis enzyme could be due to impaired
pro-tein folding, proteolytic degradation and⁄ or the lack of
a mycobacterial cofactor in the E coli expression
sys-tem Another possibility is that the mycobacterial
dehydrogenase might require a specific
post-transla-tional modification that occurs inefficiently in the
E coli host As far as we know, the specific activities
of related recombinant enzymes of plant origin have
not been reported Moreover, huge differences in the
specific activities of purified native aldonolactone
oxidoreductases, ranging from 760 mUÆmg protein)1
[17] up to 51 000 UÆmg protein)1 [12], have been
observed In particular, specific activity values
deter-mined for the native l-galactono-1,4-lactone
dehydro-genase of sweet potato [30] were 1000-fold higher than
those reported for the same enzyme by others [27], but
no explanation for this discrepancy was provided
l-Gulonolactone dehydrogenase activity could be
measured in the soluble fraction of the M bovis BCG
Copenhagen strain, and its specific activity was
compar-able to that reported for crude preparations of the
rela-ted l-galactono-1,4-lactone dehydrogenase of plant
origin [9,12,27] Altogether, our results suggest that the
mycobacterial enzyme could operate efficiently in vivo
Other proteomic studies have demonstrated that the
Rv1771 protein is relatively abundant in the M bovis
BCG strain [28] and also in M tuberculosis H37Rv,
especially in the cell envelope fraction [29] Indeed,
the Rv1771 protein contains a potential
transmem-brane helix (amino acids 205–227), as predicted by
the tmpred program (http://www.ch.embnet.org/
software/TMPRED_form.html) (Fig 2) Thus, like all
the known aldonolactone oxidoreductases, the Rv1771
protein may be membrane-associated, in agreement
with the enzyme behavior in the presence of a detergent
In contrast to the related aldonolactone oxidases
[13,16,19], but similar to the plant
l-galactono-1,4-lac-tone dehydrogenases (EC 1.3.2.3) [12], the
mycobacteri-al enzyme does not use molecular oxygen as an electron
acceptor, and has dehydrogenase activity (Table 1;
Fig 3B) Interestingly, a dehydrogenase activity of the
rat l-gulono-1,4-lactone oxidase was reported in the
early literature [26,31], but the activity was not studied
further Nowadays, the l-gulono-1,4-lactone oxidase enzymes are considered exclusively as oxidases, the reaction products of which are, paradoxically, L-AA and hydrogen peroxide [8] Dehydrogenase-to-oxidase conversion is well known for another antioxidant (uric acid)-producing enzyme, xanthine oxidoreductase [32] Perhaps a similar molecular mechanism might be responsible for the dehydrogenase-to-oxidase switch of mammalian l-gulono-1,4-lactone oxidase proteins and play a role in the metabolism of L-AA
We showed that in vitro both cytochrome c (Km4.7 lm) (Fig 3B) and phenazine methosulfate (Table 1) can serve as electron acceptors for the
l-gulono-1,4-lactone dehydrogenase of M tuberculosis Remarkably, the phenazine methosulfate acceptor was even more efficient than cytochrome c at saturation (Table 1) Phenazines, ‘secondary metabolites’ of cer-tain soil and pathogenic bacteria, are redox-active, flavin-like low-molecular-weight compounds that can produce ROS and play a role in quorum sensing and biofilm formation in Pseudomonas aeruginosa lung infection [33] It is possible, therefore, that an unknown phenazine-like, low-molecular-weight compound might serve as an endogenous electron acceptor for the Rv1771 dehydrogenase of M tuberculosis
Despite the presence of l-gulono-1,4-lactone dehy-drogenase activity in the M bovis BCG Copenhagen strain, ascorbic acid could not be detected in the M bo-visBCG and M tuberculosis cells grown in vitro, either because its concentrations were below the detection limit or because of the lack of the l-gulono-1,4-lactone substrate in these cells l-Gulono-1,4-lactone can be formed by the C1 reduction of d-glucuronic acid or
d-glucurono-3,6-lactone [8] An NADPH-dependent
d-glucurono-3,6-lactone reductase activity is present in cellular extracts of M bovis BCG (B A Wolucka, unpublished results) and could supply l-gulono-1,4-lac-tone for the dehydrogenase reaction In the animal pathway for vitamin C, free d-glucuronic acid is derived from UDP-d-glucuronate either directly by the recently proposed abortive reaction catalyzed by a UDP-glucuronosyl transferase [34] or via some poorly characterized hydrolytic steps [8] UDP-Glucuronate,
in turn, is synthesized from glucose by a UDP-glucose dehydrogenase Mycobacterium tuberculosis possesses a gene encoding a putative UDP-glucose dehydrogenase (Rv0322) that is necessary for UDP-glucuronic acid formation Moreover, the pathogen synthesizes some unknown d-glucuronate-containing glycoconjugates, as demonstrated by immunochemical methods [35], and therefore must express UDP-glucu-ronosyltransferase(s) Accordingly, a complete pathway for vitamin C synthesis, similar to the animal route
Trang 7[8,34], may exist in M tuberculosis but operate only in
specific conditions Examples of differential regulation
of gene expression and metabolic reprogramming in
M tuberculosisare known [36,37] Some earlier steps in
a pathway for ascorbic acid might be inducible, e.g
during the intracellular growth of the pathogen in its
host This could explain the absence of the
l-gulono-1,4-lactone dehydrogenase reaction product in the
M tuberculosiscells grown in vitro
The Rv1771 l-gulono-1,4-lactone dehydrogenase of
M tuberculosisis a specific enzyme for the biosynthesis
of L-AA As far as we know, this is the first report of a
specific biosynthetic enzyme for vitamin C in bacteria
To detect related aldonolactone dehydrogenases⁄
oxidases in other bacterial genomes, we used the rat
l-gulono-1,4-lactone oxidase as a query sequence in
blastsearches of the protein database These searches
retrieved, for a limited number of bacterial species,
additional putative aldonolactone oxidase orthologs
that display significant sequence identity (about 30%)
with the rat l-gulono-1,4-lactone oxidase protein, and
are closely related to the known and predicted
l-gulono-1,4-lactone oxidase-like proteins of animals,
plants and fungi (Fig 6) Surprisingly, an important
number of bacterial species that contain a vitamin C
biosynthetic gene belong to the Actinomycetales
[M tuberculosis, M bovis, Thermobifida fusca,
Streptomy-ces coelicolor and Streptomyces avermitilis (NP_823585)]
It is worth noting that members of the Actinomycetales
(Streptomyces verticillus and Saccharothrix mutabilis)
possess orthologs of another vitamin C-related enzyme,
namely the plant GDP-mannose-3¢,5¢-epimerase [38]
Ancestral soil-based organisms might therefore play a
role in horizontal transfer of vitamin C-related genes
In the process of microbial adaptation, horizontal gene
transfer is essential for the dissemination and assembly
of detoxification pathways that can form part of
genomic islands and have both pathogenicity and
degradation functions [39] Remarkably, several
l-gul-ono-1,4-lactone oxidase-positive bacteria are known
pathogens [M tuberculosis, M bovis, Burkholderia
cepacia, Bacillus anthracis (NP_654628) and
Pseudo-monas aeruginosa (NP_254014)] that infect human
lungs On the other hand, photosynthetic cynobacteria
that were largely believed to be able to synthesize
vita-min C do not, apparently, contain l-gulono-1,4-lactone
oxidase homologs, with the exception of Nostoc
puncti-forme These surprising findings raise important
ques-tions about the role of aldonolactone oxidoreductases
in prokaryotic organisms and the evolution of vitamin
C biosynthetic pathways in general
What could be the physiologic role of the Rv1771
protein in M tuberculosis? Mycobacterium tuberculosis
has coevolved with its human host, and may persist for years in a strange symbiosis known as latent infection According to World Health Organisation estimates (http://www.who.int/mediacentre/factsheets/ fs104/en/), about one-third of the world’s population
is infected with M tuberculosis but only 5–10% of the infected persons will develop active disease The Rv1771 gene is apparently not essential, because transposon mutants of the gene could be obtained [40] The gene is well conserved within the M tuber-culosis complex, except for the M bovis BCG Pasteur (1173P2) strain, which lost the gene due to the dele-tion of chromosomal region RD14 [41] Ironically, whereas the pathogen’s ortholog is well conserved, the l-gulono-1,4-lactone oxidase gene of tuberculosis-prone species, such as humans and guinea pigs, is nonfunctional because of mutations accumulated dur-ing evolution [8] These facts strongly suggest that the l-gulonolactone dehydrogenase of M tuberculosis could play a role in virulence, pathogenesis and⁄ or survival of the parasite within its host In agreement
Fig 6 Sequence relationship between L -gulono-1,4-lactone dehy-drogenase of M tuberculosis and previously identified or predicted
L -gulono-1,4-lactone oxidase-like proteins The unrooted neighbor-joining (N-J) tree was generated (http://align.genome.jp) on the basis of the amino acid sequences of proteins that show at least 30% identity with the rat L -gulono-1,4-lactone oxidase The acces-sion numbers of the sequences used were: M tuberculosis
L -gulono-1,4-lactone dehydrogenase, NP_216287; Streptomyces coelicolor, NP_629980; Thermobifida fusca, ZP_00059445; Oceano-bacillus iheyensis, NP_692632; Bacillus cereus, NP_830486; Burkholderia cepacia, ZP_00218082; Saccharomyces cerevisiae
D -arabinono-1,4-lactone oxidase (ALO), P54783; Candida albicans
D -arabinono-1,4-lactone oxidase (ALO), O93852; Neurospora crassa, Q7SGY1; Gibberella zeae, XP_388870; Arabidopsis thaliana L -galac-tono-1,4-lactone dehydrogenase (GLDH), At3g47930; Arabidopsis thaliana putative L -gulono-1,4-lactone dehydrogenase, At2g46740; Sus scrofa L -gulono-1,4-lactone oxidase (GLO), Q8HXWO; Rattus norvegicus L -gulono-1,4-lactone oxidase (GLO), P10867.
Trang 8with this, a deficiency in d-arabinono-1,4-lactone
oxidase (ALO1), which catalyzes the last step in the
biosynthesis of erythroascorbic acid in yeasts, resulted
in attenuated virulence of the Candida albicans
mutant [42] If synthesized by the Rv1771
l-gulono-1,4-lactone dehydrogenase, L-AA may represent a
novel weapon in the antioxidative arsenal of the
pathogen at least in some, although still unknown,
stages of M tuberculosis infection Interestingly, the
promoter of the cell wall catalase-peroxidase⁄ NADH
oxidase [43] gene katG of M tuberculosis, which is
important for virulence and for the activation of the
antimycobacterial prodrug isoniazid, is induced by
ascorbic acid [44] Moreover, M tuberculosis possesses
an ascorbic acid-dependent isomerase that converts
a-acetohydroxyacids to the corresponding a-ketoacids
in the pathway for branched-chain amino acids [45]
These observations suggest that L-AA could act in
M tuberculosis as a modulator of gene expression
and an enzyme cofactor, in addition to its possible
antioxidant function
In summary, the l-gulono-1,4-lactone
dehydroge-nase of M tuberculosis is a new and distinct member
of the family of l-gulono-1,4-lactone dehydrogenase⁄
oxidases that have been characterized up to now, and
the first example of a specific, vitamin C biosynthetic
enzyme of bacterial origin Further studies will be
necessary to elucidate the role of the Rv1771
l-gulono-1,4-lactone dehydrogenase in M tuberculosis
infection
Experimental procedures
Chemicals
GST affinity and StrataClean resins were obtained from
Stratagene (Madison, WI) l-Gulono-1,4-lactone,
cyto-chrome c from horse heart (oxidized form), phenazine
methosulfate and 2,6-dichloroindophenol were purchased
from Sigma-Aldrich (St Louis, MO) All reagents were of
analytical grade
Plasmid construction
The ORF corresponding to the mycobacterial Rv1771
l-gulonolactone dehydrogenase (1287 bp) was PCR
ampli-fied from the genomic DNA of M tuberculosis H37Rv
Oligonucleotide primers were designed with attB1 or attB2
sites for insertion into the Gateway donor vector
pDONR201 (Invitrogen, Gaithersburg, MD) by
homolog-ous recombination A sequence (GATGACGACGACAAG)
corresponding to the enterokinase cleavage site (DDDDK)
was included within the forward primer immediately
upstream of the start codon (ATG) Primers with the fol-lowing sequences were synthesized by Proligo (Paris, France): 5forGulox (forward), 5¢-GGGGACAAGTTT GTACAAAAAAGCAGGCTTCGATGACGACGACAAG ATGAGCCCGATATGGAGTAATTGGCCT-3¢; and 3rev-Gulox (reverse), 5¢-GGGGACCACTTTGTACAAGAAA GCTGGGTCTCAGGGACCGAGAACGCGCCGGGTGT A-3¢ The PCR product was cloned into the pDONR201 vector, and the resulting plasmid, pENTR_Rv1771, was used to transfer the gene sequence into pDEST15 (Invitro-gen) (GST tag N-terminal fusion) by means of homologous recombination The pDEST15_Rv1771 (GST fusion) plas-mid obtained was used for expression of the protein in
E coliBL21(DE3) cells
Heterologous expression of the recombinant
L-gulono-1,4-lactone dehydrogenase Escherichia coli cells carrying the pDEST15_Rv1771 plas-mid were grown at 37C to an optical density of 0.8 at
600 nm in 200 mL of culture volume, and then IPTG was added to a final concentration of 0.1 mm for induction, and the fusion protein was produced for 1 h or 3 h, as indica-ted The cells were washed, resuspended in three volumes of
100 mm phosphate buffer (pH 7.3) containing 0.1% Triton X-100, 1 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride and 20% glycerol, and sonicated After centrifuga-tion at 13 000 g for 15 min at 4C on an Eppendorf 5402 centrifuge (Eppendorf, Hamburg, Germany), the superna-tant was loaded onto a GST affinity column
GST affinity chromatography of the recombinant
L-gulono-1,4-lactone dehydrogenase of
M tuberculosis
A crude extract containing the GST-tagged dehydrogenase was applied to a 1 mL GST affinity column equilibrated with 50 mm phosphate buffer (pH 7.3) containing 0.1% Triton X-100, 1 mm phenylmethanesulfonyl fluoride and 20% glycerol (buffer A) The column was washed with 15 volumes of buffer A, and the recombinant dehydrogenase was eluted with three volumes of 10 mm glutathione (reduced form) in buffer A Fractions containing the recom-binant dehydrogenase were pooled For measurements of the l-gulono-1,4-lactone dehydrogenase activity, glutathi-one and dithiothreitol were removed by gel filtration of the pooled GST affinity fractions on a prepacked NAP-25 column (Amersham Pharmacia Biotech, Uppsala, Sweden)
Enterokinase cleavage of the GST-tagged
L-gulono-1,4-lactone dehydrogenase
In order to remove the GST tag, an aliquot of the affinity-purified recombinant dehydrogenase was incubated for 24 h
Trang 9at 22C with 2 units of enterokinase (Stratagene) in
500 lL (final volume) of 20 mm Tris⁄ HCl buffer (pH 8.0)
containing 50 mm NaCl and 2 mm CaCl2 One unit of
enterokinase is the amount of enzyme required to cleave
100 lg of the CBP-EK-JNK fusion protein (Stratagene) to
90% completion at 21C in 16 hours
Concentration of protein fractions on
Strataclean beads
To pooled GST affinity fractions, 10 lL of Strataclean
resin suspension was added After overnight incubation at
4C, samples were centrifuged at 13 000 g for 5 min at
room temperature on a Microfuge 18 centrifuge
(Beckman-Coulter, Fullerton, CA), and the supernatants discarded
The adsorbed proteins were recovered by heating the beads
in three volumes of the five-times concentrated sample
buf-fer for 5 min at 100C After centrifugation at 13 000 g for
5 min at room temperature on a Microfuge 18 centrifuge
(Beckman-Coulter), the concentrated proteins were
ana-lyzed by SDS⁄ PAGE
Preparation of crude enzyme extracts from
M bovis BCG
The M bovis BCG strain Copenhagen cultures were
grown to mid-exponential phase in Middlebrook 7H9
medium containing ADC enrichment (Becton Dickinson,
San Jose, CA) at 37C, without agitation Cells were
collec-ted by centrifugation at 1500 g for 15 min at 4C, on a
Sorvall RC5B plus centrifuge (Sorvall, Ashville, NC),
resus-pended in 100 mm phosphate buffer (pH 7.3) containing
1 mm phenylmethanesulfonyl fluoride, and disrupted by
so-nication Unbroken cells were removed by centrifugation at
500 g for 15 min at 4C, and the supernatant was further
centrifuged at 25 000 g as described above The obtained
soluble extract and the insoluble cell envelope fraction,
pre-viously resuspended in the extraction buffer, were used
for measurements of l-gulono-1,4-lactone dehydrogenase
activity
Assay ofL-gulono-1,4-lactone dehydrogenase
The reaction mixture (1 mL) contained 25 mm
l-gulono-1,4-lactone, 0.121 mm cytochrome c and an aliquot of the
affinity-purified dehydrogenase in 50 mm phosphate buffer
(pH 7.3) Because dithiothreitol and glutathione interfered
with the enzyme assay, they were removed by gel filtration
prior to measurements l-Gulono-1,4-lactone
dehydro-genase activity was measured spectrophotometrically at
550 nm by following the l-gulono-1,4-lactone-dependent
reduction of cytochrome c [46] When indicated, 2.5 mm
phenazine methosulfate was used as a direct electron
accep-tor in the presence of 100 lm 2,6-dichloroindophenol, and
the decrease in absorbance at 610 nm due to the reduction
of 2,6-dichloroindophenol was measured, as described [47]
PAGE Proteins were separated by SDS⁄ PAGE, using 10% mini-gels and the buffer system described by Laemmli [48] Gels were stained with Coomassie Brilliant Blue R-250
Immunoblotting Samples fractionated by SDS⁄ PAGE were transferred to a nitrocellulose membrane by electroblotting (Bio-Rad, Her-cules, CA) according to the manufacturer’s protocol Mem-branes were incubated with goat anti-GST IgG (1 : 1000 dilution; Amersham Pharmacia Biotech), and antibody binding was detected using anti-goat IgG conjugated to alkaline phosphatase (1 : 5000 dilution; Sigma-Aldrich) and the 5-bromo-4-chloro-3-indolyl-phosphate⁄ nitroblue tetra-zolium reagent (Promega, Madison, WI)
MS MALDI quadrupole TOF (MALDI Q-TOF) MS analysis
of in-gel-digested protein bands was performed on a Q-TOF Ultima Global mass spectrometer equipped with a MALDI source (Micromass, Waters Corporation, Milford, MA), as described [49]
Protein determination Protein concentration was determined by the method of Bradford [50], using BSA as standard
Ascorbic acid determination Mycobacterial cells were extracted with 5% m-phosphoric acid [51] or 5% perchloric acid [52], as described Ascorbic acid was measured by the HPLC method [51] by using an Alliance separation module equipped with an M2996 pho-todiode array detector and the empower chromatography software (Waters Corporation)
Acknowledgements
We wish to thank Dr P Jungblut (Max Planck Institute for Infection Biology, Berlin) for a gift of the M bovis BCG strain Copenhagen We thank Virginie Imbault for technical assistance in MS This work was partially supported by the Fonds de la Recherche Scientifique Me´dicale, Belgium (convention no 3.4.626.05.F to BAW) DC is Research Associate at the Fonds National de la Recherche Scientifique (FNRS)
Trang 101 Schwager J & Schulze J (1998) Modulation of
interleu-kin production by ascorbic acid Vet Immunol
Immuno-pathol 64, 45–57
2 Margittai E, Banhegyi G, Kiss A, Nagy G, Mandl J,
Schaff Z & Csala M (2005) Scurvy leads to endoplasmic
reticulum stress and apoptosis in the liver of guinea
pigs J Nutr 135, 2530–2534
3 Dhar-Mascareno M, Carcamo JM & Golde DW
(2005) Hypoxia–reoxygenation-induced mitochondrial
damage and apoptosis in human endothelial cells are
inhibited by vitamin C Free Radic Biol Med 38, 1311–
1322
4 Fischer H, Schwarzer C & Illek B (2004) Vitamin C
controls the cystic fibrosis transmembrane conductance
regulator chloride channel Proc Natl Acad Sci USA
101, 3691–3696
5 Brown LAS & Jones DP (1997) Antioxidant action of
vitamin C in the lung In Vitamin C in Health and
Dis-ease(Packer L & Fuchs J, eds), pp 265–278 Marcel
Dekker, New York
6 May JM, Huang J & Qu ZC (2005) Macrophage uptake
and recycling of ascorbic acid: response to activation by
lipopolysaccharide Free Radic Biol Med 39, 1449–1459
7 Valpuesta V & Botella MA (2004) Biosynthesis of
l-ascorbic acid in plants: new pathways for an old
antioxidant Trends Plant Sci 9, 573–577
8 Nishikimi M & Yagi K (1996) Biochemistry and
mole-cular biology of ascorbic acid biosynthesis Subcell
Biochem 25, 17–39
9 Wolucka BA & Van Montagu M (2003) GDP-mannose
3¢,5¢-epimerase forms GDP-l-gulose, a putative
interme-diate for the de novo biosynthesis of vitamin C in plants
J Biol Chem 278, 47483–47490
10 Shigeoka S, Nakano Y & Kitaoka S (1979) Some
prop-erties and subcellular localization of l-gulono-c-lactone
dehydrogenase in Euglena gracilis z Agric Biol Chem
43, 2187–2188
11 Mapson LW & Breslow E (1958) Biological synthesis of
ascorbic acid: l-galactono-c-lactone dehydrogenase
Biochem J 68, 395–406
12 Ostergaard J, Persiau G, Davey MW, Bauw G & Van
Montagu M (1997) Isolation of a cDNA coding for
l-galactono-c-lactone dehydrogenase, an enzyme
involved in the biosynthesis of ascorbic acid in plants
Purification, characterization, cDNA cloning, and
expression in yeast J Biol Chem 272, 30009–30016
13 Kiuchi K, Nishikimi M & Yagi K (1982) Purification
and characterization of l-gulonolactone oxidase from
chicken kidney microsomes Biochemistry 21, 5076–
5082
14 Loewus FA (1999) Biosynthesis and metabolism of
ascorbic acid in plants and of analogs of ascorbic acid
in fungi Phytochemistry 52, 193–210
15 Bleeg HS & Christensen F (1982) Biosynthesis of ascor-bate in yeast Purification of l-galactono-1,4-lactone oxidase with properties different from mammalian
l-gulonolactone oxidase Eur J Biochem 127, 391– 396
16 Huh WK, Kim ST, Yang KS, Seok YJ, Hah YC & Kang SO (1994) Characterisation of d-arabinono-1,4-lactone oxidase from Candida albicans ATCC 10231 Eur J Biochem 225, 1073–1079
17 Nishikimi M, Noguchi E & Yagi K (1978) Occurrence
in yeast of l-galactonolactone oxidase which is similar
to a key enzyme for ascorbic acid biosynthesis in animals, l-gulonolactone oxidase Arch Biochem Biophys
191, 479–486
18 Huh WK, Lee BH, Kim ST, Kim YR, Rhie GE, Baek
YW, Hwang CS, Lee JS & Kang SO (1998) D-Ery-throascorbic acid is an important antioxidant molecule
in Saccharomyces cerevisiae Mol Microbiol 30, 895– 903
19 Wilkinson SR, Prathalingam SR, Taylor MC, Horn D
& Kelly JM (2005) Vitamin C biosynthesis in trypano-somes: a role for the glycosome Proc Natl Acad Sci USA 102, 11645–11650
20 Koshizaka T, Nishikimi M, Ozawa T & Yagi K (1988) Isolation and sequence analysis of a complementary DNA encoding rat liver l-gulono-c-lactone oxidase, a key enzyme for l-ascorbic acid biosynthesis J Biol Chem 263, 1619–1621
21 Sugisawa T, Ojima S, Matzinger PK & Hoshino T (1995) Microbial production of l-ascorbic acid from
d-sorbitol, l-sorbose, l-gulose, and l-sorbosone by Ketogulonicigenium vulgare DSM 4025 Biosci Biotech-nol Biochem 59, 190–196
22 Nakagawa H, Asano A & Sato R (1975) Ascorbate-synthesizing system in rat liver microsomes II A peptide-bound flavin as the prosthetic group of l-gulono-c-lac-tone oxidase J Biochem (Tokyo) 77, 221–232
23 Nishikimi M, Kiuchi K & Yagi K (1977) Detection of
l-gulono-c-lactone oxidase on SDS-polyacrylamide gels
by the fluorescence of its covalently bound flavin FEBS Lett 81, 323–325
24 Kito M, Ohishi N & Yagi K (1991) Micro-determina-tion of l-gulono-c-lactone oxidase activity Biochem Int
24, 131–135
25 Yabuta Y, Yoshimura K, Takeda T & Shigeoka S (2000) Molecular characterization of tobacco mitochon-drial l-galactono-c-lactone dehydrogenase and its expression in Escherichia coli Plant Cell Physiol 41, 666–675
26 Eliceiri GL, Lai EK & McCay PB (1969) Gulonolactone oxidase Solubilization, properties, and partial purifica-tion J Biol Chem 244, 2641–2645
27 Imai T, Karita S, Shiratori G, Hattori M, Nunome T, Oba K & Hirai M (1998) l-Galactono-c-lactone dehydrogenase from sweet potato: purification and