(Received for publication, August 14, 1995; and in revised form, October 6, 1995)
From the
The comparison of the deduced amino acid sequences of plant and
animal sulfotransferases (ST) has allowed the identification of four
well conserved regions, and previous experimental evidence suggested
that regions I and IV might be involved in the binding of the
cosubstrate, 3`-phosphoadenosine 5`-phosphosulfate (PAPS). Moreover,
region IV is homologous to the glycine-rich phosphate binding loop
(P-loop) motif known to be involved in nucleotide phosphate binding in
several protein families. In this study, the function of amino acid
residues within these two regions was investigated by site-directed
mutagenesis of the plant flavonol 3-ST. In region I, our results
identify Lys as critical for catalysis, since replacement
of this residue with alanine resulted in a 300-fold decrease in
specific activity, while a 15-fold reduction was observed after the
conservative replacement with arginine. Photoaffinity labeling of K59R
and K59A with [
S]PAPS revealed that Lys
is not required for cosubstrate binding. However, the K59A mutant
had a reduced affinity for 3`-phosphoadenosine 5`-phosphate
(PAP)-agarose, suggesting that Lys
may participate in the
stabilization of an intermediate during the reaction. In region IV, all
substitutions of Arg
resulted in a marked decrease in
specific activity. Conservative and unconservative replacements of
Arg
resulted in weak photoaffinity labeling with
[
S]PAPS and the R276A/T73A and R276E enzymes
displayed reduced affinities for PAP-agarose, suggesting that the
Arg
side chain is required to bind the cosubstrate. The
analysis of the kinetic constants of mutant enzymes at residues
Lys
, Gly
, and Lys
allowed to
confirm that region IV is involved in cosubstrate binding.
Sulfotransferases catalyze the transfer of a sulfonate group
from an activated nucleotide donor, 3`-phosphoadenosine
5`-phosphosulfate (PAPS), ()to the appropriate alcoholic or
phenolic hydroxyl groups of acceptor substrates. In contrast with plant
tissues in which STs have yet to be assigned a particular function, in
mammals these enzymes play an important role in the detoxification of
xenobiotics and endogenous metabolites, as the presence of a sulfate
group increases water solubility of hydrophobic molecules and
facilitates their excretion. In addition, STs are involved in the
metabolic pathways of biologically active molecules, such as steroid
hormones and neurotransmitters. In that case, it is generally well
established that sulfate conjugation of such compounds is important to
modulate their biological activity(1) . Research conducted to
elucidate the role of flavonoid sulfation in plants has resulted in the
isolation and biochemical characterization of four position-specific
STs which are involved in the stepwise formation of flavonol
polysulfates(2, 3) . The plant flavonol 3- and 4`-STs
exhibit strict specificity for position 3 of flavonol aglycones and 4`
of flavonol 3-sulfates, and cDNA clones encoding these two enzymes were
isolated and characterized(4) . In a recent investigation, we
constructed a series of hybrid enzymes by the substitution of protein
segments between the flavonol 3- and 4`-STs. Analysis of substrate
preference of the resulting chimeric proteins allowed the
identification of a domain located in the central portion of these
enzymes that is responsible for both substrate and position
specificities(5) .
Progress in understanding the structure-function relationship of STs has been limited by the fact that their three-dimensional structure has not yet been resolved. However, a large number of cDNA clones coding for STs of different specificities have been isolated from various organisms. The comparison of the deduced amino acid sequences of ST enzymes of plant and animal origin has revealed significant homology, and four well conserved regions have been identified(4, 6) . These conserved regions could participate in shared functions of these enzymes, such as cosubstrate binding or specifying the proper folding for catalysis.
Two of the conserved regions of STs represent almost uninterrupted blocks of sequence identity. The conserved region I is located in the N-terminal portion of STs and its sequence is YPKSGT(T/N)W (Fig. 1). It is interesting to note that this motif is also present in two bacterial STs which, otherwise, exhibit very weak general homology with their eukaryotic functional homologs(14, 15) . Recently, affinity labeling experiments with a nucleotide analog allowed the identification of two labeled amino acid residues located in the N-terminal part of the rat hepatic aryl ST IV(16) . However, it is unlikely that these amino acid residues are involved in PAPS binding, since they are not conserved among all cloned STs, but their proximity to the amino acids of region I suggests that the latter may interact with the cosubstrate.
Figure 1: Schematic representation of the conserved regions among STs and amino acid sequence alignments of the conserved regions I and IV. The backbone corresponds to a protein of 311 amino acids (pFST3). The amino acid sequence alignment includes the flavonol 3- and 4`-STs (FST3 and FST4) with Flaveria bidentis ST-like cDNA(7) , human liver hydroxysteroid ST (HSST)(8) , rat hydroxysteroid ST (RHST)(9) , rat minoxidil ST (RMST)(10) , human aryl ST (HAST)(11) , human dehydroepiandrosterone ST (HDST)(12) , and human estrogen ST (EST)(13) . Also aligned for the conserved region I are the bacterial STs amino acid sequences encoded by nodH from Rhizobium meliloti(14) and the ORF4 of the avrD locus from Pseudomonas syringae pv. tomato(15) . The boxes indicate residues common to the nine eukaryotic STs. Position numbers refer to the flavonol 3-ST sequence. Dots indicate positions of the amino acid residues that were modified in this study. The alignment was obtained using LINEUP in the GCG package, except for NodH and ORF4, which were aligned visually.
The conserved region IV, on the other hand, is located in the
C-terminal portion of STs and its sequence is
RK(G/A)XXGDWK(N/T)XFT. Regions sharing homology with
this motif have been identified in the nonhomologous, membrane-bound N-heparan sulfate ST (17) and in adenosine
phosphosulfate kinases(18) . The motif
GXXGXXK present in region IV has been proposed to act
as a ``PAPS-binding site'' because of its homology with the
consensus sequence GXXXXGK, described as the glycine-rich
phosphate binding loop (P-loop) known to be involved in nucleotide
phosphate binding in a number of enzymes(19, 20) .
Crystal structures of adenylate kinase, p21 and
F
-ATPase bound to substrate analogs have revealed that the
P-loop wraps around the phosphate groups of the nucleotide and that the
side chain of the invariant lysine is positioned to make contact with
the
- and
-phosphates of ATP or
GTP(21, 22, 23) . The critical role of the
lysine residue in substrate binding has been confirmed by the results
of affinity labeling and site-directed mutagenesis studies of several
enzymes(20) . In addition, it has been suggested that the
lysine side chain is directly involved in transition state
stabilization of adenylate kinase(24, 25) .
The involvement of region IV in PAPS binding has recently been suggested by the results of a site-directed mutagenesis study of the guinea pig estrogen ST(26) . However, since this study made use of triple mutants within the region, the contribution of discrete positions to cosubstrate binding could not be evaluated. In order to further characterize the structure and function relationship of STs, we have modified amino acid residues located in the conserved regions I and IV of the flavonol 3-ST by site-directed mutagenesis. In this paper, we describe the results of experiments which allowed to identify residues present in these two regions that are important for cosubstrate binding and catalysis.
In order to identify amino acids involved in cosubstrate
binding and catalysis, we modified the amino acid Lys
within the conserved region I, and Arg
,
Lys
, Gly
, and Lys
within the
conserved region IV by site-directed mutagenesis of the flavonol 3-ST (Fig. 1). Conservative replacement of basic amino acids was
sought, as well as nonconservative replacement with either alanine, in
order to minimize structural alteration and to prevent the formation of
hydrogen bonds (35) , or with glycine, which eliminates side
chain interactions. The recombinant wild-type and mutant enzymes were
expressed in E. coli and were readily purified from bacterial
supernatants by affinity chromatography on nickel-agarose. The
recombinant proteins migrated at a distance corresponding to the
predicted molecular mass of 35 kDa and were at least 95% pure, as
evaluated from SDS-PAGE (data not shown). The levels of expression and
stability in solution of the different proteins were comparable,
suggesting that no drastic modifications of the tertiary structure were
induced by the mutations. Although the recombinant flavonol 3-ST has 12
additional amino acids at its N-terminal, the kinetic properties of the
nickel-agarose-purified enzyme were similar to those of the enzyme
purified from the plant (Table 1)(30) . However, the K
for quercetin was slightly higher (0.45 versus 0.20 µM for the plant enzyme) whereas the K
for PAPS was similar (0.22 µMversus 0.18 µM). The k
value of 1.43 s
was representative of the
value of 1.86 s
obtained with the purified plant
enzyme (Table 1).
In region I, replacement of Lys with alanine resulted in an approximately 300-fold reduction in
specific activity. Replacement of Lys
with arginine, on
the other hand, resulted in an approximately 15-fold reduction in
specific activity and a 2-fold decrease of the K
for PAPS, while the K
for quercetin was
unchanged (Table 1). In region IV, all mutations of Arg
resulted in a pronounced decrease of catalytic activity. The cDNA
encoding the mutant R276A was found to contain an additional mutation
changing the unconserved Thr
residue to alanine. However,
since the results obtained with the double mutant R276A/T73A were
consistent with those obtained for other substitutions of
Arg
, they were included in this study. Mutants R276K and
R276A/T73A displayed approximately 5000- and 500-fold lower specific
activities as compared with that of the recombinant wild-type enzyme,
while mutant R276E had no detectable activity (Table 1).
Conservative replacement with arginine of Lys
and
Lys
, the latter corresponding to the invariant lysine in
the P-loop motif, had no significant effect on the k
or the K
for both substrates.
Nonconservative replacement of Lys
and Lys
with glycine gave rise to mutant proteins having a similar
decrease in kcat (approximately 2-4-fold) and increase of the K
for PAPS (approximately 6-9-fold) (Table 1). The K
for quercetin for both
mutants was similar to that of the wild-type recombinant enzyme. The
effect on the kinetic constants of replacing Gly
with
alanine was comparable with that observed for mutants K277G and K284G (Table 1).
The binding properties of the inactive or very
weakly active mutants toward the sulfate donor were characterized by
photoaffinity labeling with [S]PAPS. UV
irradiation of a crude soluble protein extract of E. coli harboring pFST3 in the presence of 1 µM
[
S]PAPS resulted in the labeling of a protein
migrating at a position corresponding to that of the recombinant
wild-type flavonol 3-ST (Fig. 2, A and B). As
expected, the addition of a 100-fold molar excess of unlabeled PAP
completely prevented labeling of the recombinant wild-type enzyme.
Photoaffinity labeling of the K59A mutant was similar to that of the
K59R and recombinant wild type enzymes (Fig. 2, A and B), indicating that PAPS binding is not impaired in this
mutant. These results strongly suggest that although conservative and
unconservative replacements of Lys
have an impact on
catalytic activity, this residue is not required for cosubstrate
binding. In contrast, photoaffinity labeling of R276K, R276E, and
R276A/T73A resulted in similar bands of very weak intensity, supporting
the role of Arg
in PAPS binding (Fig. 2, A and B). The intensities of the photoaffinity labeled
products of K277G, G281A, and K284G were intermediate between those of
the Arg
mutants and recombinant wild-type enzyme (Fig. 2, C and D). These results are
consistent with the 5-9-fold increases of the K
for PAPS observed for these three mutants.
Figure 2:
[S]PAPS
photoaffinity labeling of the recombinant wild-type and mutant flavonol
3-STs. A, SDS-PAGE of the protein extracts of the recombinant
wild-type enzyme and of the Lys
and Arg
mutants after the photoaffinity labeling reaction. B,
autoradiograph obtained with the same protein preparations shown in A. C, SDS-PAGE of the protein extracts of the recombinant wild
type flavonol 3-ST and of the K277G, G281A, and K284G mutants after the
photoaffinity labeling reaction. D, autoradiograph obtained
with the same protein preparations shown in C. The protein
band corresponding to the flavonol 3-ST is indicated by an arrow. rF3ST, histidine-tagged recombinant wild type
flavonol 3-sulfotransferase.
To further
characterize mutants of Lys and Arg
that did
not have a sufficient level of catalytic activity for reliable kinetic
analysis, they were submitted to affinity chromatography on
PAP-agarose. The plant flavonol 3-ST is sensitive to product inhibition
by PAP, a competitive inhibitor of PAPS for the active site of the
enzyme, with a K
(0.1 µM) slightly
lower than the K
for PAPS (0.18
µM)(30) . As expected, the recombinant wild-type
flavonol 3-ST bound strongly to the PAP-agarose affinity matrix and was
eluted with 0.78 M NaCl with good reproducibility between
individual experiments. The activity profile of the recombinant
wild-type enzyme coincided with the elution profiles determined by
monitoring the absorbance at 280 nm (Fig. 3) and by
immunodetection of the purified fractions (Fig. 4). The strong
interaction of the recombinant wild-type flavonol 3-ST with the PAP
affinity matrix is similar to that previously observed with the plant
enzyme(30) .
Figure 3: Elution profile of the recombinant wild-type, K59R, and K59A enzymes following chromatography on PAP-agarose. rF3ST, histidine-tagged recombinant wild type flavonol 3-sulfotransferase.
Figure 4: Immunoblot of the PAP-agarose purified fractions of the recombinant wild-type, K59A, R276K, R276E, and R276A/T73A enzymes with anti-F. chloraefolia flavonol 3-sulfotransferase (F3ST) antibodies. The letter E refers to the desalted extract, and FT refers to the flow-through of the column. Numbers indicate the PAP-agarose-purified fractions. rF3ST, histidine-tagged recombinant wild type flavonol 3-sulfotransferase.
The mutant proteins retained the ability to
bind to PAP-agarose, although significant differences were observed in
the salt concentration required for their elution. Mutant K59A eluted
at 0.56 M NaCl, indicating a weaker affinity for PAP than the
recombinant wild-type flavonol 3-ST ( Fig. 3and Fig. 4).
The elution profile of the K59R mutant was similar to the recombinant
wild-type enzyme, reflecting the fact that it displays only a minor
change in the K for PAPS. Mutant R276K also eluted
at the same salt concentration as the recombinant wild-type enzyme ( Fig. 4and 5). In contrast, mutants R276E and R276A/T73A eluted
at a lower salt concentration of 0.64 and 0.66 M,
respectively. The reduction in affinity for PAP-agarose observed with
mutant R276E, as compared with R276A/T73A, could be due to a charge
repulsion between the phosphate groups of PAP and the carboxyl group of
the glutamyl side chain. These results strongly suggest that both
Lys
and Arg
are involved in PAP binding
through ionic interactions.
Valid interpretation of the affinity
chromatography data requires a prior demonstration that the affinity
for PAP-agarose is specific. The following evidence suggests that the
interaction with PAP-agarose is highly specific: 1) PAP-agarose
affinity chromatography has already been applied to the purification of
several STs, and it has been shown that they can be specifically eluted
from the support by the addition of PAP or PAPS at a concentration of 1
mM or less (36, 37, 38) . 2) To test
whether nonspecific ionic interactions could contribute in a
significant way to the affinity for PAP-agarose, control experiments
were performed with mutants involving a change to the net charge of the
enzyme. Mutant E101K, that displays no change in kinetic constants
(data not shown), eluted at the same salt concentration as the
recombinant wild-type enzyme, indicating that the introduction of a
positive charge did not enhance binding to the negatively charged
chromatographic support. 3) Mutants K277G and K284G, having
6-9-fold increases of the K for PAPS, showed
only slight reductions of affinity for PAP-agarose compared to the
recombinant wild-type flavonol 3-ST, eluting respectively at 0.75 and
0.77 M NaCl. Since these reductions are much smaller than
those observed for K59A, R276A/T73A, and R276E, they cannot be
interpreted only by the loss of a positive charge on the mutant
proteins. Taken together, these results support the hypothesis that the
interaction of the enzyme with PAP-agarose is specific. However, the
technique does not allow the accurate detection of small differences in
affinity for the immobilized ligand.
In this study, the function of conserved residues within regions I and IV of STs was investigated (Fig. 1). Several important features justify their choice as targets for site-directed mutagenesis. First, they represent almost uninterrupted blocks of sequence identity present in all eukaryotic cytoplasmic STs. Also, a secondary structure algorithm predicts that both regions form loop structures frequently associated with the formation of active sites. Finally, region IV is homologous to the P-loop involved in nucleotide phosphate binding in several enzymes(17) .
The involvement
of region I in cosubstrate binding is suggested by the recent affinity
labeling of the amino acids Lys and Cys
of
the rat hepatic aryl ST IV by the nucleotide analog ATP
dialdehyde(16) . However, these amino acids are only conserved
among the members of the phenol and estrogen ST families, and
site-directed mutagenesis of the corresponding cysteine residue to
serine in the human liver phenol ST has revealed that it is not
involved in substrate binding or catalysis, but is important for the
thermal stability of the enzyme(39) . In view of the proximity
of the affinity labeled amino acids to region I, Zheng et al.(16) proposed that the latter might be involved in the
interaction with the cosubstrate. Our results identify Lys
within this region as critical for catalysis, since replacement of this
amino acid with alanine produces a pronounced decrease in specific
activity. The results of photoaffinity labeling studies clearly
indicate that Lys
is not required for PAPS binding, since
K59A is labeled to a similar extent as the K59R and recombinant
wild-type enzymes, suggesting that this residue acts as a catalyst in
the flavonol 3-ST. In the enzyme-PAPS complex, the Lys
side chain may be too distant to interact with the cosubstrate,
but when a longer arginine side chain is introduced at this position,
it may interact weakly with the sulfate donor. This is consistent with
the results of the affinity labeling experiments, and the small but
significant reduction of the K
for PAPS of the
K59R mutant, that was reproduced in several independent experiments. On
the other hand, the reduced affinity of K59A for PAP-agarose compared
with that of the K59R and recombinant wild-type enzymes suggests that
Lys
binds a phosphate group of PAP through an ionic
interaction, indicating that this residue may stabilize the leaving
group of the reaction. In the absence of a proposed catalytic mechanism
for STs, we can only speculate that the role of Lys
may be
to stabilize an intermediate and/or to lower the activation energy of a
transition state.
The region IV of STs shares sequence homology with
the phosphate binding loop involved in nucleotide phosphate binding in
several protein families. However, a distinctive feature of region IV
is the presence of additional conserved residues on both sides of the
segment homologous to the P-loop motif. The function of residues
specific to the ST motif (Arg and Lys
) and
of residues homologous to those of the P-loop motif (Gly
and Lys
) was investigated. All substitutions of
Arg
resulted in a dramatic decrease in specific activity,
and the results of photoaffinity labeling studies suggest that this
residue is involved in the formation of the enzyme-PAPS complex.
Furthermore, the interaction is specific for the arginine side chain as
demonstrated by the drastic reduction in catalytic activity of the
R276K mutant. In addition, Arg
is also involved in the
binding of the product of the reaction as suggested by the reduction in
affinity for PAP-agarose of mutants R276E and R276A/T73A. The
participation of Arg
in product binding suggests that it
may also be involved in catalysis. These results are in agreement with
the previous finding by chemical modification with phenylglyoxal that
one arginine residue is required for catalysis in the rat liver phenol
ST (40) . We cannot exclude the possibility that structural
alterations are induced by the substitutions at Lys
and
Arg
, but the normal affinities for PAPS of the Lys
mutants and the normal affinity for PAP-agarose observed for
R276K suggest that there is no major change of the tertiary structure
in these mutants.
The results of the kinetic analysis and
photoaffinity labeling of mutants K277G, G281A, and K284G support the
involvement of region IV in cosubstrate binding. Although mutations at
these positions have a moderate impact on the formation of the
enzyme-PAPS complex and on the catalytic activity of the enzyme, they
may participate with Arg in the binding of the
cosubstrate. In a recent mutational study of the guinea pig estrogen
ST, it was found that the replacement with alanine of the amino acids
corresponding to Gly
, Gly
, and Lys
of the flavonol 3-ST resulted in a triple mutant with no
catalytic activity that could not be photoaffinity labeled with
[
S]PAPS(26) . Our results on Gly
and Lys
suggest that the absence of catalytic
activity in this mutant is due to the cumulative effects of the three
substitutions on cosubstrate binding. Alternatively, these residues may
play a role in maintaining the proper conformation of the loop region.
The effects on the catalytic constants observed with mutants K277G,
G281A, and K284G may result from subtle structural changes affecting
the position of the Arg
side chain.
This study
represents an important step toward an understanding of catalysis in
STs. Our results confirm that the conserved region IV of STs and the
P-loop motif are functionally related in that both are involved in the
binding of nucleotide cosubstrates. In view of the absolute
conservation of the amino acids Lys and Arg
in all cloned eukaryotic cytoplasmic STs, it is likely that the
results presented here can be extended to all members of this class of
enzymes. Other electrophilic loci may be needed in addition to
Arg
to stabilize the negatively charged groups of the
cosubstrate, especially since PAPS is not bound by STs as a chelate
complex with a divalent cation, and a residue acting as a base catalyst
may be required to abstract a proton from the hydroxyl group of the
acceptor substrate to activate it for nucleophilic attack at the
sulfuryl group. To address these aspects of catalysis by the
sulfotransferases, the construction and analysis of site-directed
mutants at other conserved residues of the flavonol 3-ST are presently
under progress.