COMMUNICATION
Site-selected Mutagenesis of a Conserved Nucleotide Binding
HXGH Motif Located in the ATP Sulfurylase Domain of
Human Bifunctional 3'-Phosphoadenosine 5'-Phosphosulfate
Synthase*
K. V.
Venkatachalam
,
Hirotoshi
Fuda
,
Eugene V.
Koonin§, and
Charles A.
Strott
¶
From the
Section on Steroid Regulation, Endocrinology
and Reproduction Research Branch, NICHD and the § National
Center for Biotechnology Information, National Library of Medicine,
National Institutes of Health, Bethesda, Maryland 20892-4510
 |
ABSTRACT |
3'-Phosphoadenosine-5'-phosphosulfate (PAPS)
synthase is a bifunctional protein consisting of an
NH2-terminal APS kinase and a COOH-terminal ATP
sulfurylase. Both catalytic activities require ATP; the APS kinase
domain involves cleavage of the
-
phosphodiester bond of ATP,
whereas the ATP sulfurylase domain involves cleavage of the
-
phosphodiester bond of ATP. Previous mutational studies have suggested
that
-
phosphodiesterase activity involves a highly conserved
NTP-binding P-loop motif located in the adenosine-5'-phosphosulfate kinase domain of PAPS synthases. Sequence alignment analysis of PAPS
synthases and the superfamily of TagD-related nucleotidylyltransferases revealed the presence of a highly conserved HXGH motif in
the ATP sulfurylase domain of PAPS synthases, a motif implicated in the
-
phosphodiesterase activity of cytidylyltransferases. Thus, site-selected mutagenesis of the HXGH motif in the ATP
sulfurylase domain of human PAPS synthase (amino acids 425-428) was
performed to examine this possibility. Either H425A or H428A mutation
produced an inactive enzyme. In contrast, a N426K mutation resulted in increased enzymatic activity. A G427A single mutant resulted in only a
modest 30% reduction in catalytic activity, whereas a G427A/H428A double mutant produced an inactive enzyme. These results suggest an
important role for the HXGH histidines in the ATP
sulfurylase activity of bifunctional PAPS synthase and support the
hypothesis that the highly conserved HXGH motif found in
the ATP sulfurylase domain of PAPS synthases is involved in ATP binding
and
-
phosphodiesterase activity.
 |
INTRODUCTION |
In the course of sulfonation, inorganic sulfate must be activated
prior to being transferred to an acceptor molecule (1), and the
activated sulfate molecule is 3'-phosphoadenosine 5'-phosphosulfate (PAPS).1 The activation of
inorganic sulfate to form PAPS results from the concerted action of two
enzymes (1). The first step is catalyzed by ATP sulfurylase
(ATP:sulfate adenylyltransferase, EC 2.7.7.4) and involves the reaction
of inorganic sulfate with ATP to form adenosine 5'-phosphosulfate (APS)
and inorganic pyrophosphate. This reaction results in the formation of
a high energy phosphoric-sulfuric acid anhydride bond that is the
chemical basis for sulfate activation (2). The second step involves APS
reacting with another molecule of ATP to form PAPS and ADP and is
catalyzed by APS kinase (ATP:adenylylsulfate 3'-phosphotransferase, EC
2.7.1.25). ATP sulfurylase and APS kinase cloned from bacteria, Fungi,
yeast, and plants are found on separate polypeptide chains; however, in
mammalian species, the marine worm, and the fruit fly, gene fusion has
occurred, and the two enzymes are integral to bifunctional PAPS
synthase. It has been clearly established that the
NH2-terminal region of human (h) PAPS synthase constitutes
the APS kinase domain, whereas the ATP sulfurylase domain is located in
the COOH-terminal portion of this bifunctional protein (3). That is,
the NH2-terminal 268 amino acids of hPAPS synthase (623 amino acids) when overexpressed and purified functions independently as
an APS kinase (3), whereas the overexpressed and purified COOH-terminal
405 amino acids of hPAPS synthase functions independently as an ATP
sulfurylase.2
A highly conserved P-loop NTP-binding motif
(GXXGXGK(S/T)) is present within the
NH2-terminal APS kinase domain of human (3), guinea
pig,2 mouse (4), marine worm (5), and Drosophila
(6) PAPS synthase. The P-loop motif is highly conserved among
NTP-binding proteins where it is involved in binding the phosphate
moiety and in the cleavage of the
-
phosphodiester bond of NTP
(7, 8). It was recently reported that site-selected mutagenesis of the
P-loop motif in mouse PAPS synthase markedly impaired APS kinase
activity (9). The latter finding would be in keeping with the
hypothesis that the P-loop motif located near the NH2 terminus of PAPS synthase is involved in cleavage of the
-
phosphodiester bond of ATP and transfer of the terminal phosphoryl
group of ATP to the 3'-hydroxyl position of adenosine
5'-phosphosulfate.
In contrast to the NH2-terminal APS kinase domain of PAPS
synthase, the COOH-terminal ATP sulfurylase domain does not and would
not necessarily be expected to contain a classical P-loop motif for ATP
binding. The reason being that although the COOH-terminal ATP
sulfurylase domain of PAPS synthase binds ATP as does the NH2-terminal domain, the ultimate fate of the bound ATP is
not the same. That is, APS kinase catalyzes the removal of the terminal
-phosphate for transfer to the acceptor molecule, APS, whereas ATP
sulfurylase catalyzes the removal of the
-
diphosphate (inorganic pyrophosphate) of ATP and condensation of the formed AMP with inorganic
sulfate to form APS. It might, therefore, be expected that the latter
function would require a different type of nucleotide-binding site, one
that would function as an
-
phosphodiesterase rather than a
-
phosphodiesterase. Inspection of the data base search (10)
output for the COOH-terminal domain of hPAPS synthase revealed a
HXGH motif that is conserved throughout the PAPS synthase
family. This motif has been previously characterized as the signature of a large family of (predicted) nucleotidylyltransferases, prototyped by the Bacillus subtilis cytidylyltransferase TagD; all
functionally characterized proteins of this family cleave the
-
phosphate bond of an NTP (11).
In this paper, we report that site-selected mutagenesis of the
HXGH motif implicates this motif in the ATP sulfurylase
reaction of bifunctional hPAPS synthase, a finding consistent with the concept that the HXGH sequence is involved in ATP binding
and
-
phosphodiesterase activity.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Radionucleotides [
-35S]ATP for
DNA sequencing, inorganic [35S]SO4 (1300 Ci/mmol), and [35S]PAPS for enzyme assays were purchased
from NEN Life Science Products. Oligonucleotides were obtained from
Life Technologies, Inc. and Gene Probe Technology (Gaithersburg, MD).
Site-directed mutagenesis kit was obtained from Stratagene (La Jolla,
CA). Version-2 sequencing kit was obtained from U. S. Biochemical
Corp. Agarose was purchased from FMC BioProducts (Rockland, ME) and
polyethyleneimine cellulose (PEI)-TLC plates were purchased from Merck.
Overexpression of Human PAPS Synthase and Mutant
Constructs--
Wild type full-length hPAPS synthase
(GenBankTM accession number U53447) and mutant hPAPS
synthase constructs were amplified by PCR using primers designed to
contain BamHI restriction sites, cloned into
BamHI digested pET-19b vectors containing a proprietary 122-base pair NcoI-NdeI cassette (Veritas,
Potomac, MD) encoding the calmodulin-binding site of calcineurin
followed by a histidine tag and an enterokinase cleavage site, and used
for transformation of Escherichia coli essentially as
described previously (3). Plasmids were used for transformation of
DH-5
competent E. coli cells by the CaCl2
method. Transformants were isolated and miniprepped, and plasmids were
sequenced for correct orientation of the initiator codon with respect
to the T7 promoter sequence. The pET-19b vectors containing the correct
inserts were isolated and used for transformation of expression host
cells from Stratagene (BL21-DE3 plyz).
Site-selected Mutagenesis--
The conserved purported
NTP-binding motif (HXGH) located in the COOH-terminal region
of human PAPS synthase was subjected to mutational analysis. Thus,
amino acid substitutions were carried out in the HNGH sequence (amino
acids 425-428). Site-selected mutagenesis was performed according to
Stratagene's quik change mutagenesis kit. For example,
oligonucleotides (54 nucleotides) containing the respective base
substitutions were synthesized. Employing the wild type hPAPS synthase
expression vector plasmid pET-19b, mutations were performed by PCR
using the substituted primer and Pfu DNA polymerase. Thermal
cycle phases consisted of either 12 cycles (single mutation) or 16 cycles (double mutation) of denaturation at 94 °C for 30 s,
annealing at 55 °C for 1 min, and extension at 68 °C for 12 min.
After PCR, the methylated parent template plasmid was digested with the
DpnI restriction enzyme, and the circular dsDNA was used to
transform XL1-Blue supercompetent cells. Colonies were isolated,
miniprepped, and sequenced. Verified mutant plasmids were used to
transform pLyz BL21-DE3 overexpression bacteria that are deficient in
proteases. Plasmids were isolated and sequenced to verify the presence
of the proper mutation.
Preparation of Bacterial Cell Extracts--
Colonies were grown
in LB broth containing ampicillin to an A595 nm
of ~0.5, and IPTG was added to a final concentration of 1 mM to induce expression. Induction was carried out for
3 h. Cells were collected by centrifugation, the pellets were
resuspended in 150 µl of lysis buffer (20 mM Tris-HCl, pH
7.5, containing 50 mM KCl, 1 mM dithiothreitol,
10% glycerol, and 1.2 mg/ml lysozyme), and the cell suspension was
transferred to a microcentrifuge tube. The original tube was washed
with 100 µl of lysis buffer, the wash was added to the cell
suspension, cell lysis was carried out by incubation at 25 °C for 7 min, and lysates were centrifuged for 15 min at 10,000 × g at 4 °C.
Purification of Histidine Fusion Proteins--
Wild type hPAPS
synthase and mutant proteins were purified using nickel columns.
Bacteria were grown in LB broth to an A595 nm of ~0.5, at which time 1 mM IPTG was added to induce
expression as noted above. Cells were collected by centrifugation and
lysed using lysis buffer as before, and the clear extract was mixed with 1 ml of slurry of nitrilotriacetic acid agarose (Qiagen, Chatsworth, CA). The contents were gently rotated on a wheel at 4 °C
for 1.5 h. The contents were then poured into small columns and
washed with buffer A (20 mM Tris-HCl, pH 8.0, 100 mM KCl, 10% glycerol, 1 mM dithiothreitol)
until the A280 nm was less than 0.01. The
columns were subsequently washed with six column volumes of buffer A
containing 10 mM imidazole and eluted with 1 ml of buffer A
containing 100 mM imidazole.
PAPS Synthase Assay--
Enzyme activity was determined in a
total volume of 10 µl consisting of 3 µl of sample, 3 µl of
reaction buffer (150 mM Tris-HCl, pH 8.0, 50 mM
KCl, 15 mM MgCl2, 3 mM EDTA, and 45 mM dithiothreitol), 1 µl of 50 mM ATP, and 3 µl of inorganic [35S]SO4 (~3.4 µCi).
Reactions were carried out for 30 min at 37 °C and stopped by
placing the reaction tubes in boiling water for 5 min. Aliquots (1 µl) were transferred to PEI-TLC plates and developed using 0.9 M LiCl as the solvent system. Following chromatography, the
PEI-TLC plates were dried and exposed overnight to x-ray film (Eastman
Kodak Co.). The respective spots for PAPS, APS, and SO4
were excised, and the radioactivity was determined by liquid scintillation.
Western Blot Analysis--
Expressed purified proteins (30 µl
from 1 ml peak fractions) were resolved by SDS-polyacrylamide gel
electrophoresis using 12% polyacrylamide gels obtained from Novex,
Inc. (San Diego, CA) and electroblotted onto Immobilon-P membranes from
Millipore (Bedford, MA). Membranes were soaked in a solution of 0.1%
bovine serum albumin in phosphate-buffered saline containing 0.1%
Tween 20 (blocking buffer) for 45 min by gentle shaking, after which they were exposed to primary antibody (1:1000), which had been preadsorbed with E. coli extract (0.5 mg/ml), for 1 h.
The primary antibody was generated against a COOH-terminal peptide
fragment of human PAPS synthase as previously reported (3). Blots were washed three times with wash buffer (1× phosphate-buffered saline with
0.01% Tween 20) and incubated with the secondary antibody in blocking
buffer (1:5000) for an additional 1 h. Finally, the membranes were
subjected to three washes in wash buffer and two additional washes in
reaction buffer (0.1 M Tris-HCl, pH 8.0). The blots were
developed by color reaction according to the manufacturer's instructions (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD).
Amino Acid Sequence Alignment Analyses--
Searches of the
protein sequence data base (the nonredundant data base at the NCBI)
were performed using the gapped BLAST program, the position-specific
iterative BLAST (PSI-BLAST), and the pattern-hit initiated BLAST
(PHI-BLAST) programs (10, 12). Multiple alignments were constructed
using either the Clustal W program (13) or the MACAW program (14,
15).
 |
RESULTS |
Amino Acid Sequence Analyses
Initially, the presence of the HX(G/A)H motif that is
strictly conserved in the COOH-terminal, ATP sulfurylase domain of the PAPS synthases as well as ATP sulfurylases was noticed by visual inspection of the data base search outputs and multiple alignments of
this domain (data not shown). Data base searches started with the PAPS
synthase sequences because the queries failed to detect significant
similarity to any members of the TagD superfamily, although limited
similarity to some of them was detectable when the search was performed
using the PHI-BLAST program that combined BLAST analysis with the
detection of the HX(G/A)H pattern (12). By contrast, in a
search initiated with the sequence of the nucleotidylyltransferase domain of the NadR protein from Mycobacterium tuberculosis
using the PSI-BLAST program (10), members of the PAPS synthase
superfamily were retrieved at a statistically significant level
(p < 0.01) within 8-10 search iterations (10, 16).
Although quite subtle (11% identity on a 189-amino acid overlap), the
alignment between NadR and PAPS synthases encompassed almost the whole
nucleotidylyltransferase domain (data not shown). Multiple alignment
analysis using the MACAW program (14, 15) showed that not only the
HX(G/A)H but also the surrounding hydrophobic positions that
form the basis of a predicted
-loop-
structural unit were
conserved in the PAPS synthases and the TagD superfamily (Fig.
1).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 1.
Multiple alignment of the predicted NTP
binding site. Analysis using the MACAW program involved selected
bifunctional PAPS synthases and ATP sulfurylases (upper
block) and TagD superfamily nucleotidylyltransferases (lower
block). The conserved HX(G/A)H motif is shown by
reverse type, and additional conserved residues are shown by
bold type. In the consensus line, h indicates
hydrophobic residues and s indicates small residues.
PAPS SYN, PAPS synthase; ATP SUL, ATP
sulfurylase; CITC, acetate:SH-citrate lyase ligase;
RIBF, riboflavin kinase/FMN adenylyltransferase;
TAGD, glycerol-3-phosphate cytidylyltransferase;
PANC, pantothenate synthase; CTPT,
CTP-phosphocholine cytidylyltransferase; NADR, NAD operon
regulator; KDTB, lipopolysaccharide core biosynthesis
protein; DROME, Drosophila melanogaster;
YEAST, Saccharomyces cerevisiae;
CHLRE, Chlamydomonas reinhardtii;
ARATH, Arabidopsis thaliana; SYNSP,
Synechocytis sp.; ECOLI, E. coli;
MYCPN, Mycoplasma pneumoniae; BACSU,
B. subtilis; MYCTU, M. tuberculosis.
The first column of numbers represents the gene
identification; the second column of numbers
represents the amino acid residue location.
|
|
Expression of Wild Type and Mutant hPAPS Synthase
Western Blotting--
Expression of partially purified wild type
and mutant protein preparations was clearly demonstrated (Fig.
2). The level of expression of the mutant
construct N426A was consistently higher than the other constructs in
three independent experiments, whereas the level of expression for all
other constructs was nearly equal. In a mock control, i.e.
vector without a hPAPS synthase insert, no protein expression was
observed (Fig. 2).

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 2.
Immunoblot of bacterially expressed wild type
and mutant hPAPS synthase proteins purified by column
chromatography. Overexpression of plasmid constructs, preparation
of cell extracts, purification of fusion proteins, and Western analysis
were carried out as described under "Experimentral Procedures." The
primary antibody was developed against a COOH-terminal-specific peptide
of hPAPS synthase. Samples applied were: lane 1, wild type;
lanes 2-6, mutants H425A, N426K, G427A, H428A, and
G427A/H428A, respectively; lane 7, mock control. Protein
applications for lanes 1-7 were: 0.27, 0.3, 0.1, 0.1, 0.15, 0.2, and 0.5 µg/30 µl of sample volume, respectively.
|
|
Catalytic Activity--
Vectors (wild type and mutants) not under
the control of promoter induction by IPTG, as well as mock controls,
exhibited no demonstrable catalytic activity.
Relative PAPS synthase specific activities in cellular extracts for
wild type hPAPS synthase, the mutant constructs, and the mock control
are presented in Fig. 3. The mutant
constructs H425A, H428A, and G427A/H428A exhibited only background
activity similar to that of the mock control. On the other hand, the
mutant construct N426K formed 185.8 pmol PAPS/min·mg, which was about
2-fold higher than the activity of 96.8 pmol PAPS/min·mg demonstrated
by wild type hPAPS synthase. The G427A mutant formed 72.2 pmol
PAPS/min·mg, a value slightly less than wild type.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 3.
Relative overall PAPS synthase specific
activities in cellular extracts of wild type (WT),
mutant constructs, and mock control. Overexpression of plasmid
constructs, preparation of cell extracts, and PAPS synthase assays were
carried out as described under "Experimentral Procedures."
|
|
 |
DISCUSSION |
Sequence analysis has shown the relatedness of the
nucleotidylyltransferase superfamily and TagD and revealed a connection between this category of enzymes and the class I aminoacyl-tRNA synthetases whose structure is known (11). Based on crystal structure
for aminoacyl-tRNA synthetases, structural predictions for the
nucleotidylyltransferases mapped a conserved HXGH motif to a
position analogous to the location of the ATP-binding HIGH motif in the
tRNA synthetases (11). Thus, a structural and functional link was
proposed for the TagD superfamily and the class I aminoacyl-tRNA synthetases with the HXGH motif in the cytidylyltransferase
superfamily functioning as a NTP-binding site analogous to the HIGH
motif serving as an ATP-binding site for the tRNA synthetases.
Interestingly, the HXGH motif is completely conserved in
cloned bifunctional PAPS synthase proteins, i.e. the marine
worm (5), mouse (4, 17), human (3), guinea pig,2 and fruit
fly (6) as well as the ATP sulfurylases from fungi (18, 19), plants
(20, 21), and yeast (22). Importantly, the ATP sulfurylase domain of
bifunctional PAPS synthase is located in the COOH-terminal region of
the protein (3), and it is in this segment of PAPS synthase that the
HXGH motif is located.
A unifying functional feature of the TagD nucleotidylyltransferase
superfamily and the aminoacyl-tRNA synthetases is their
-
phosphodiesterase activity. It has been proposed that the conserved
HXGH motif in this superfamily is involved in NTP binding (23), an hypothesis strengthened by recent mutational analyses (24).
Our finding that mutating either H425A or H428A of the HXGH
motif in hPAPS synthase produced an inactive enzyme is comparable with
the results of similar mutations carried out with CTP:phosphocholine cytidylyltransferase (24). The G427A mutation of hPAPS synthase resulted in approximately a 30% reduction in overall enzymatic activity, a finding also comparable with a similar mutation carried out
with CTP:phosphocholine cytidylyltransferase (24). Thus, as with the
TagD superfamily, mutational studies with hPAPS synthase support the
proposition that the highly conserved HXGH motif in the ATP
sulfurylase domain of PAPS synthases is involved in NTP binding.
Crystal structures of aminoacyl-tRNA synthetases indicate that the HIGH
sequence is located in the first loop of a
-
-
unit, which
allows the imidazole nitrogens to form hydrogen-binding contacts with
the phosphate oxygens of ATP (11). Mutational studies of the
HXGH motif in CTP:phosphocholine cytidylyltransferase also
implicate the involvement of similar contacts with CTP (24). It follows
that the results of the mutational studies involving the
HXGH motif in hPAPS synthase should be analogously
interpreted. Whereas the H425A and H428A mutants of hPAPS synthase
produced inactive enzymes, only a modest reduction (~30%) in PAPS
synthase activity was noted with the G427A mutant compared with the
wild type enzyme. On the other hand, the N426K mutant yielded an
increase in PAPS synthase activity, suggesting that the
-NH3 group of lysine creates a more efficient enzyme
than the amide NH2 of asparagine, perhaps by creating
enhanced hydrogen contact with ATP.
During PAPS formation by bifunctional PAPS synthase (ATP
sulfurylase/APS kinase) two molecules of ATP are required, one for each
reaction. Site-selected mutagenesis of a P-loop motif
(GXXGXGK(T/S)) located in the
NH2-terminal, APS kinase domain of mouse PAPS synthase markedly impaired APS kinase activity (9), a finding in keeping with
the purported role of the NTP-binding P-loop motif in the cleavage of
the
-
phosphodiester bond of ATP and transfer of the terminal
phosphoryl group to the 3'-hydroxyl position of APS. In contrast to the
NH2-terminal, APS kinase domain, the COOH-terminal, ATP
sulfurylase domain of PAPS synthase, acting as an
-
phosphodiesterase during the formation of APS from ATP and inorganic
sulfate, requires a different type of NTP-binding site. Based on the
results of site-selected mutagenesis of hPAPS synthase, we propose that
the domain homologous to the TagD superfamily nucleotidylyltransferases and in particular, the completely conserved HXGH motif
located in the ATP sulfurylase domain of all bifunctional PAPS
synthases fulfills such a function.
 |
ACKNOWLEDGEMENT |
We gratefully acknowledge the contributions of
Anna M. Leung, who worked as a summer student in the laboratory.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Bldg. 49, Rm.
6A36, NIH, Bethesda, MD 20892-4510. Tel.: 301-496-3025; Fax:
301-496-7435; E-mail: chastro{at}box-c.nih.gov.
The abbreviations used are:
PAPS, 3'-phosphoadenosine 5'-phosphosulfate; hPAPS, human PAPS; APS, adenosine 5'-phosphosulfate; PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-
-D-galactopyranoside; PEI, polyethyleneimine cellulose.
2
K. V. Venkatachalam and C. A. Strott,
unpublished data.
 |
REFERENCES |
-
Gregory, J. D.,
and Robbins, P. W.
(1960)
Annu. Rev. Biochem.
29,
347-364[CrossRef]
-
Leyh, T. S.
(1993)
Crit. Rev. Biochem. Mol. Biol.
28,
515-542[Abstract]
-
Venkatachalam, K. V.,
Akita, H.,
and Strott, C. A.
(1998)
J. Biol. Chem.
273,
19311-19320[Abstract/Free Full Text]
-
Li, H.,
Deyrup, A.,
Mensch, J. R., Jr.,
Domowicz, M.,
Konstantinidis, A. K.,
and Schwartz, N. B.
(1995)
J. Biol. Chem.
270,
29453-29459[Abstract/Free Full Text]
-
Rosenthal, E.,
and Leustek, T.
(1995)
Gene (Amst.)
165,
243-248[CrossRef][Medline]
[Order article via Infotrieve]
-
Jullien, D.,
Crozatier, M.,
and Kas, E.
(1997)
Mech. Dev.
68,
179-186[CrossRef][Medline]
[Order article via Infotrieve]
-
Walker, J. E.,
Saraste, M.,
Runswick, M. J.,
and Gay, N. J.
(1982)
EMBO J.
1,
945-951[Medline]
[Order article via Infotrieve]
-
Saraste, M.,
Sibbald, P. R.,
and Wittinghofer, A.
(1990)
Trends Biochem. Sci.
15,
430-434[CrossRef][Medline]
[Order article via Infotrieve]
-
Deyrup, A. T.,
Krishnan, S.,
Cockburn, B. N.,
and Schwartz, N. B.
(1998)
J. Biol. Chem.
273,
9450-9456[Abstract/Free Full Text]
-
Altschul, S. F.,
Madden, T. L.,
Schaffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402[Abstract/Free Full Text]
-
Bork, P.,
Holm, L.,
Koonin, E. V.,
and Sander, C.
(1995)
Proteins
22,
259-266[Medline]
[Order article via Infotrieve]
-
Zhang, Z.,
Schaffer, A. A.,
Miller, W.,
Madden, T. L.,
Lipman, D. J.,
Koonin, E. V.,
and Altschul, S. F.
(1998)
Nucleic Acids Res.
26,
3986-3990[Abstract/Free Full Text]
-
Thompson, J. D.,
Higgins, D. G.,
and Gibson, T. J.
(1994)
Nucleic Acids Res.
22,
4673-4680[Abstract]
-
Schuler, G. D.,
Altschul, S. F.,
and Lipman, D. J.
(1991)
Proteins
9,
180-190[Medline]
[Order article via Infotrieve]
-
Neuwald, A. F.,
Liu, J. S.,
and Lawrence, C. E.
(1995)
Protein Sci.
8,
1618-1632
-
Altschul, S. F.,
and Koonin, E. V.
(1998)
Trends Biochem. Sci.
23,
444-447[CrossRef][Medline]
[Order article via Infotrieve]
-
Kurima, K.,
Warman, M.,
Krishnan, S.,
Domowicz, M.,
Krueger, R. C.,
Deyrup, A.,
and Schwartz, N. B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8681-8685[Abstract/Free Full Text]
-
Foster, B. A.,
Thomas, S. M.,
Mahr, J. A.,
Renosto, F.,
Patel, H. C.,
and Segel, I. H.
(1994)
J. Biol. Chem.
269,
19777-19786[Abstract/Free Full Text]
-
Borges-Walmsley, M. I.,
Turner, G.,
Bailey, A. M.,
Brown, J.,
Lehmbeck, J.,
and Clausen, I. G.
(1995)
Mol. Gen. Genet.
247,
423-429[Medline]
[Order article via Infotrieve]
-
Leustek, T.,
Murillo, M.,
and Cervantes, M.
(1994)
Plant Physiol.
105,
897-902[Abstract/Free Full Text]
-
Murillo, M.,
and Leustek, T.
(1995)
Arch. Biochem. Biophys.
323,
195-204[CrossRef][Medline]
[Order article via Infotrieve]
-
Cherest, H.,
Kerjan, P.,
and Surdin-Kerjan, Y.
(1987)
Mol. Gen. Genet.
210,
307-313[Medline]
[Order article via Infotrieve]
-
Veitch, D. P.,
and Cornell, R. B.
(1996)
Biochemistry
35,
10743-10750[CrossRef][Medline]
[Order article via Infotrieve]
-
Veitch, D. P.,
Gilham, D.,
and Cornell, R. B.
(1998)
Eur. J. Biochem.
255,
227-234[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.