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J Bacteriol, June 1998, p. 3218-3221, Vol. 180, No. 12
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The MTCY428.08 Gene of
Mycobacterium tuberculosis Codes for
NAD+ Synthetase
Rita
Cantoni,1
Manuela
Branzoni,1
Monica
Labò,1
Menico
Rizzi,1,2 and
Giovanna
Riccardi1,*
Dipartimento di Genetica e Microbiologia,
Università di Pavia, 27100 Pavia,1
and
Dipartimento di Scienza e Tecnologia del Farmaco,
Università di Torino, 10125 Torino,2 Italy
Received 23 February 1998/Accepted 10 April 1998
 |
ABSTRACT |
The product of the MTCY428.08 gene of
Mycobacterium tuberculosis shows sequence homology with
several NAD+ synthetases. The MTCY428.08 gene
was cloned into the expression vectors pGEX-4T-1 and pET-15b.
Expression in Escherichia coli led to overproduction of
glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the MTCY428.08 gene
of M. tuberculosis is the structural gene for
NAD+ synthetase.
| |
TEXT |
NAD+ is a vital and
ubiquitous coenzyme involved in a variety of biochemical processes,
comprising not only redox reactions but also DNA repair, DNA
recombination, and ADP protein ribosylation (7, 17).
NAD+ biosynthesis is therefore crucial in all living
organisms and can be pursued through a de novo pathway or through a
pyridine nucleotide salvage pathway (7, 18).
The ubiquitous enzyme NAD+ synthetase (EC 6.3.5.1)
catalyzes the last step in NAD+ biosynthesis transforming
deamido-NAD+ into the final product NAD+ by a
two step reaction: (i) first it activates deamido-NAD+
through the formation of an adenylated NAD+ in the presence
of ATP and magnesium, and then (ii) an ammonia molecule attacks the
adenylated intermediate leading to NAD+ (15).
NAD+ synthetase belongs to the amidotransferase family,
whose members are responsible for the transfer of the amide
nitrogen of glutamine on different substrates in a large
number of biochemical processes (20).
Amidotransferases are characterized by the presence of two
distinct domains, which may either belong to the same polypeptide chain
or exist as independent subunits. A glutamine amide transfer (GAT)
domain is responsible for the ability to use glutamine as nitrogen
source, whereas a synthetase/synthase domain confers the specificity
and catalyzes the transfer of ammonia to the substrate. Amidotransferases can be grouped into two families depending on their
GAT domains: (i) G-type amidotransferases having a Cys-His-Glu catalytic triad and (ii) F-type or Ntn (N-terminal nucleophile) amidotransferases possessing a conserved N-terminal cysteine
(20).
NAD+ synthetase from Bacillus subtilis has been
extensively characterized and reported to be strictly ammonia dependent
(10), even if the existence of a yet unknown second subunit,
bearing the GAT domain, cannot be excluded. The three-dimensional
structure of the B. subtilis enzyme in its free form and in
complex with ATP has also been reported (12), allowing the
assessing of catalytically important residues and confirming the early
proposal that NAD+ synthetase, GMP synthetase,
asparagine synthetase, and arginosuccinate synthetase constitute a new
class of N-type ATP pyrophosphatases (ATP-PPases), characterized by the
conserved fingerprint sequence S-G-G-X-D (16).
The major role played by NAD+ synthetase in cellular
metabolism is underlined by previously reported studies on
B. subtilis mutants: all mutants with
altered NAD+ synthetase activity were shown to be severely
affected in cellular functionality (5).
The recent resurgence of drug resistance tuberculosis poses a serious
threat to the control of the desease and has caused much public concern
(4). It has been demonstrated that a lack of nicotinic acid
phosphoribosyltransferase activity in Mycobacterium tuberculosis blocks the NAD+ recycling (7).
Moreover, the use of the frontline antitubercular drug isoniazid may
deplete pools of NAD+: there is a decrease in
NAD+ of tubercule bacilli grown in the presence of
isoniazid (3). These observations make the de novo
NAD+ biosynthesis in general and NAD+
synthetase in particular possible targets for the development of new
drugs against this microorganism.
Here we demonstrate that the MTCY428.08 gene from
M. tuberculosis is the structural gene for
NAD+ synthetase.
The MTCY428.08 gene of M. tuberculosis is
the structural gene of NAD+ synthetase.
The use of the
B. subtilis NAD+ synthetase
sequence as a search probe against the sequenced M. tuberculosis genome showed significant homology with the
MTCY428.08 gene, coding for a polypeptide chain 738 residues
long. The corresponding amino acid sequence compared with nonredundant
databases at the National Center for Biotechnology Information by
using the BLAST programs (1, 2) revealed low but
significant homology with several NAD+ synthetases.
The highest similarity (P, 2.1e
21)
was with the corresponding protein from Rhodobacter
capsulatus (19). The degrees of sequence identity
between the M. tuberculosis MTCY428.08 gene product
and NAD+ synthetases from R. capsulatus and a Synechocystis sp. are, respectively, 31.4% (in a 191-residue overlap) and 29.7% (in a 182-residues overlap). The deduced amino acid sequence of the MTCY428.08
gene was aligned, by using the CLUSTAL program (8), with
those of other bacterial NAD+ synthetases, revealing a
strict conservation of residues showed to be important in catalysis
(Fig. 1) (12). The MTCY428.08
amino acid sequence contains the fingerprint sequence SGGXDST
(residues 427 to 433) proposed to be characteristic of a new family of
ATP-PPases which includes NAD+ synthetase (12,
16).
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FIG. 1.
(A) Amino acid sequence alignment of the B. subtilis, M. genitalium, R. capsulatus, and
M. tuberculosis NAD+ synthetases. The consensus
fingerprint sequence for the N-type ATP-PPases (16) is
boxed. Identical residues are indicated (asterisks). (B) Amino acids
sequence alignment (8) of the A subunit of the B. subtilis Glu-AdT and the N-terminal (N-ter) region of the M. tuberculosis NAD synthetase.
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|
The M. tuberculosis MTCY428.08 gene was cloned from cosmid
MTCY428 (11) by PCR, according to standard protocol, into
the expression vectors pGEX-4T-1 (14) and pET-15b (Novagen).
The resulting plasmids were designated pCAB1 and pBAC2, respectively. pCAB1 and pBAC2 were transformed, by electroporation, into
Escherichia coli JM105 and E. coli
BL21(DE3), respectively, according to the supplier's
instructions.
Following aerobic growth on 2× YTG medium (Pharmacia-Biotech) in the
presence of ampicillin (100 µg/ml), total cell lysates
of
E. coli JM105, containing plasmid pGEX-4T-1 or pCAB1 (the
recombinant
plasmid), were prepared 120 min after the start of
isopropyl-1-thio-
-
D-galactopyranoside
(IPTG)
induction and analyzed by sodium dodecyl
sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE).
E. coli JM105 cells transformed
with the pGEX-4T-1
vector showed high-level expression of the
26-kDa glutatione
S-transferase (GST) protein. The presence of
a 98-kDa polypeptide (the
predicted molecular mass of the
M. tuberculosis MTCY428.08
protein fused to GST) was clearly evident in extracts
of
E. coli JM105 cells harboring plasmid pCAB1 (data not shown).
After
aerobic growth on Luria-Bertani (LB) medium (
13) in the
presence of carbenicillin (200 µg/ml), total cell lysates of
E. coli BL21(DE3), containing plasmid pET-15b or pBAC2 (the
recombinant
plasmid), were prepared at various times from the start of
IPTG
induction and analyzed by SDS-PAGE. Growth at 37°C, even if the
recombinant protein was highly expressed, caused the target protein
to
accumulate mainly as inclusion bodies. In order to avoid this
undesirable effect, the incubation was carried out at 22°C without
IPTG induction (Fig.
2, lane 2). In the
latter case the His
6-tagged
protein was purified through
affinity chromatography on a Ni chelate
resin according to the
supplier's instructions (Fig.
2, lane 6).
Care was taken to avoid
oxidative conditions, maintaining a stable
concentration of
-mercaptoethanol (10 mM) through all the purification
steps.
The first 30 N-terminal residues of the
MTCY428.08
gene
product for both the GST-fused and His
6-tagged
proteins were sequenced,
confirming the expected sequence.
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FIG. 2.
SDS-PAGE analysis of the M. tuberculosis NAD
synthetase purification profile. Lanes: 1, molecular weight standards;
2, E. coli BL21(DE3)(pET-15b) lysate supernatant; 3, E. coli BL21(DE3)(pBAC2) S-30 supernatant; 4, E. coli BL21(DE3)(pBAC2) S-100 supernatant; 5, E. coli
BL21(DE3)(pBAC2) ultracentrifuged supernatant; 6, partially purified
NAD+ synthetase (arrow).
|
|
The activity of the
M. tuberculosis NAD
+
synthetase enzyme was routinely assayed with ammonia or glutamine as
nitrogen source
as previously described (
9,
21).
NAD
+ synthetase activity was assayed on crude extracts of
E. coli JM105 transformed with plasmids pGEX-4T-1 and pCAB1
or
E. coli BL21(DE3) transformed with plasmids pET-15b
and pBAC2 and on the
partially purified protein. The activity was
expressed as nanomoles
of NAD
+ synthesized per minute per
milligram of protein. The results
obtained are listed in Table
1.
The identification of the
M. tuberculosis NAD
+
synthetase was firstly made possible through sequence comparison with
other
NAD
+ synthetases, at the National Center for
Biotechnology Information.
Sequence alignment revealed the strictly
conserved sequence fingerprint
characteristic of the N-type ATP-PPase
(
16). Moreover, a careful
sequence comparison of the
B. subtilis and
M. tuberculosis enzymes,
in light
of the three-dimensional structure available for the
former, showed
a remarkable conservation of all the catalytically
relevant residues
(
12).
The definitive confirmation that the
MTCY428.08 gene codes
for NAD
+ synthetase was provided by determination of the
enzymatic activity.
When the dialyzed crude extracts of strain
JM105(pCAB1) were assayed,
the specific ammonia- or glutamine-dependent
activities were found
to be similar and roughly 15-fold greater than
that of the control
strain. The same behavior was detected for the
BL21(DE3)(pBAC2)
strain, but the specific activities were only six
times greater
than those of the control strain BL21(DE3)(pET-15b).
These low
values, even if undoubtedly confirming the presence of
NAD
+ synthetase activity, may indicate that the enzyme is
poorly expressed
in its active form. After partial purification, the
specific activity
increased to a value of 230 nmol min
1
mg of protein
1.
Another aspect clearly evidenced by the sequence alignment shown in
Fig.
1 is a striking difference in molecular dimensions
for the
different NAD
+ synthetases, ranging from about 200 residues
to 738 amino acids
in the case of
M. tuberculosis protein. A
possible explanation
of this fact is that the smaller enzymes represent
only the synthetase
subunit of the entire amidotransferase where the
GAT domain is
most likely synthesized as an independent subunit,
whereas the
bigger enzymes contain both domains on the same polypeptide
chain.
In this respect the observation that the
B. subtilis
enzyme (consisting
of 271 residues) is strictly ammonia dependent
(
10) whereas
the
M. tuberculosis enzyme
(consisting of 738 residues) is able
to use ammonia or glutamine as
nitrogen source with comparable
specific activities (this study) is in
keeping with our proposal.
Recently an extensive study on
B. subtilis Glu-tRNA
Gln amidotransferase (Glu-AdT) was
reported. Glu-AdT is a heterotrimeric
protein whose A subunit,
responsible for the amidotransferase
function in this complex enzyme,
does not have any obvious sequence
relationship either with F-type or
G-type known amidotransferases.
On the other hand, it has a peculiar
signature sequence, strictly
conserved among a family of amidases.
Therefore, it has been proposed
to have a new type of amidase-like
domain (
6).
Interestingly, we observed significant sequence homology between the
N-terminal region (residues 1 to 409) of the
M. tuberculosis NAD synthetase and the Glu-AdT A subunit (Fig.
1B).
However, the
N-terminal domain of NAD synthetase does not possess the
signature
sequence, shared by Glu-AdT and several amidases
(
6). In this
respect our finding suggests that the
N-terminal region of NAD
synthetase and the Glu-AdT A subunit may
represent two members
of a new type of GAT domain.
Our work provides the bases for future extensive characterization of
this important enzyme from
M. tuberculosis, hopefully
including a crystallographic investigation, with the final aim
of
development of new antitubercular drugs.
| |
ACKNOWLEDGMENTS |
This work was supported by National Tuberculosis Project (Istituto
Superiore di Sanità) contracts 96/D/T5696 and 96/D/T49 and by the
European Union Research project BIOMED CT-961241.
We thank S. T. Cole for providing the M. tuberculosis
cosmid Y428.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dipartimento di
Genetica e Microbiologia, Università di Pavia, via Abbiategrasso
207, 27100 Pavia, Italy. Phone: (39) 382 505 574. Fax: (39) 382 52 84 96. E-mail: Riccardi{at}ipvgen.unipv.it.
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J Bacteriol, June 1998, p. 3218-3221, Vol. 180, No. 12
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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