From the Department of Biochemistry and Molecular
Biology, Michigan State University, East Lansing, Michigan
48824-1319 and the § Center for Agricultural Molecular
Biology, Department of Plant Science, Rutgers University,
New Brunswick, New Jersey 08903-0231
Received for publication, September 7, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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The sulfolipid sulfoquinovosyldiacylglycerol is a
component of plant photosynthetic membranes and represents one of the
few naturally occurring sulfonic acids with detergent properties. Sulfolipid biosynthesis involves the transfer of sulfoquinovose, a
6-deoxy-6-sulfoglucose, from UDP-sulfoquinovose to diacylglycerol. The
formation of the sulfonic acid precursor, UDP-sulfoquinovose, from
UDP-glucose and a sulfur donor is proposed to be catalyzed by the
bacterial SQDB proteins or the orthologous plant SQD1 proteins. To
investigate the underlying enzymatic mechanism and to elucidate the
de novo synthesis of sulfonic acids in biological systems, we developed an in vitro assay for the recombinant SQD1
protein from Arabidopsis thaliana. Among different possible
sulfur donors tested, sulfite led to the formation of
UDP-sulfoquinovose in the presence of UDP-glucose and SQD1. An SQD1
T145A mutant showed greatly reduced activity. The
UDP-sulfoquinovose formed in this assay was identified by
co-chromatography with standards and served as substrate for the
sulfolipid synthase associated with spinach chloroplast membranes.
Approximate Km values of 150 µM for
UDP-glucose and 10 µM for sulfite were established for
SQD1. Based on our results, we propose that SQD1 catalyzes the
formation of UDP-sulfoquinovose from UDP-glucose and sulfite, derived
from the sulfate reduction pathway in the chloroplast.
The sulfolipid 6-sulfo- The elucidation of the reactions of sulfolipid biosynthesis by
biochemical means has been recalcitrant in the past, but recently powerful new experimental tools became available with the isolation of
sulfolipid-deficient mutants of the purple bacterium Rhodobacter sphaeroides and the cloning of the first genes encoding enzymes of
sulfolipid biosynthesis, sqdA, sqdB,
sqdC, and sqdD (5-7). The sulfonic acid
precursor giving rise to the sulfolipid head group was originally
postulated by A. A. Benson to be UDP-sulfoquinovose (UDP-SQ) (8),
and synthetic UDP-SQ was later shown to specifically stimulate
sulfolipid biosynthesis in isolated chloroplast membranes (9, 10). Only
recently, the existence of UDP-SQ in living cells has been demonstrated
using a sulfolipid-deficient mutant of R. sphaeroides that
accumulates UDP-SQ (11). Subsequently, UDP-SQ was discovered also in
other photosynthetic organisms (12). Indirect clues toward the
elucidation of sulfolipid biosynthesis could be deduced from the
sequences of putative sqd gene products. In particular, the
SQDB protein of R. sphaeroides has sequence similarity to
sugar nucleotide-modifying enzymes, and orthologous proteins in
bacteria and plants are highly conserved (13). It was suggested that
these proteins catalyze a reaction between UDP-glucose (UDP-Glc)
and a suitable sulfur donor leading to the formation of UDP-SQ (5, 14).
The SQD1 protein of Arabidopsis thaliana is an orthologue of
the bacterial SQDB proteins (15), and its crystal structure has been
elucidated (16). Recombinant SQD1 lacking the chloroplast transit
peptide has a mass of 45.5 kDa, forms a dimer, and contains a buried
active site with tightly bound NAD+ (16, 17).
Co-crystallization with UDP-Glc demonstrated directly the binding of
this presumed substrate in the active site. Furthermore, in place of
the sulfur donor, water molecules were present. Labeled sulfate is
incorporated into SQDG by isolated chloroplasts (18-20), and it seemed
likely that the sulfur donor is derived from the sulfate reduction
pathway in the chloroplast. Intermediates of this pathway, adenosine
5'-phosphosulfate (APS) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS), were indeed incorporated into SQDG by isolated chloroplasts (21). Another intermediate of the sulfate reduction pathway, sulfite,
has not been tested in this system but was shown to be incorporated
into SQDG by extracts of Chlamydomonas reinhardtii (22).
However, this reaction was linear over time and therefore thought to be
nonenzymatic. Here, we provide evidence for the SQD1-catalyzed
formation of the sulfolipid head group donor UDP-SQ from UDP-Glc and
sulfite in vitro.
Substrates--
The following sulfur-containing substrates were
used: sulfate (J. T. Baker Inc.), sulfite (Merck), sulfide (Sigma),
APS (Sigma), PAPS (Sigma), glutathione (reduced and oxidized forms)
(Sigma), and thiosulfate (Mallinckrodt Chemical Works, St. Louis, MO). Sulfoglutathione was synthesized according to Ref. 23 and purified by
TLC and visualized as described (24). Labeled
UDP-[14C(U)]glucose and
UDP-[14C(U)]galactose were purchased from American
Radiolabeled Chemicals, Inc. (St. Louis, MO). Labeled
UDP-[35S]sulfoquinovose was prepared using the
sqdD mutant of R. sphaeroides and analyzed by TLC
as described (11). Labeled [35S]APS was prepared from
[35S]PAPS (PerkinElmer Life Sciences) by phosphatase
treatment (25). Unless otherwise specified below, common buffer
ingredients were obtained from general commercial sources.
Assay Conditions for SQD1 and HPLC Analysis--
Recombinant
A. thaliana SQD1 protein excluding the transit peptide was
purified on Ni2+-nitrilotriacetic acid columns (Qiagen,
Valencia, CA) as described by Essigmann et al. (15). The
column was eluted with 200 mM imidazole, which was
subsequently removed by use of a Millipore Corp. (Bedford, MA)
Ultrafree 4 concentrator. The SQD1 protein was stored in 20% glycerol,
300 mM NaCl, and 25 mM
NaH2PO4 (pH 7.5) at Sulfolipid Synthase Assay--
Chloroplasts were isolated from
spinach (locally available produce) according to Rossak et
al. (11). Chlorophyll was quantified by the method of
Lichtenthaler (26). The assay consisted of 35 µg of chlorophyll with
10,000 dpm of radioactive compound U2 in 50 µl of
reaction buffer (50 mM Tricine/KOH, 30 mM
MgCl2, pH 7.5). For control purposes, galactolipid
synthesis was monitored using UDP-[14C(U)]galactose (0.4 µM, 1200 Bq nmol Site-directed Mutagenesis--
An SQD1 mutant derivative was
constructed by PCR mutagenesis employing a method of Ito et
al. (27). Threonine 145 was changed to alanine using the primers
5'-ATACTCACCCATCGCCCCAAGTTTTAC-3' and 5'-ATCACCATCACGGCTCCCGTGTTAT-3'.
PCR products were cloned into pPCR-Script Amp SK(+) (Stratagene, La
Jolla, CA), and mutant plasmids were sequenced at the MSU sequencing
facility. The mutant open reading frame was inserted into pQE30
(Qiagen, Valencia, CA), expressed in Escherichia coli, and
the protein was purified as described (15).
In Vitro Reaction of UDP-Glc and Sulfite Mediated by SQD1--
An
enzyme assay was developed to measure the conversion of UDP-Glc to
UDP-SQ as predicted for SQD1 activity. SQD1 protein was estimated to be
at least 95% pure by gel analysis (Fig.
1). To broadly analyze the reaction
mixtures for substrates and reaction products, we employed an HPLC
system optimized for the separation of sugar nucleotides. Sample
through-put was limited, in particular because columns had to be
regenerated extensively after few runs. Therefore, we also explored
different TLC systems and filter-based assays to separate substrates
and products with the goal of processing large sample numbers in
parallel. However, none of the alternative procedures we designed, thus
far, were satisfactory with regard to sample recovery or
reproducibility. Because of the limitation in sample through-put, data
points shown are representative for at least three different
independent experiments instead of averages of multiple repeats in a
single experiment. We routinely used labeled UDP-Glc as tracer for the
radioassay. Incubation of the SQD1 protein with labeled UDP-Glc in a
simple Tris buffer, as described under "Experimental Procedures,"
resulted in the formation of two compounds with unique retention times
as compared with UDP-Glc (Fig. 2,
A and B). At the end of the assay period, the protein was routinely denatured to release any products or
intermediates still bound in the active site of the protein. Filtration
of the reaction mixture using Amicon filters (Mr
cut-off 10,000; Millipore) without denaturation revealed that 77% of
compound U2 (Fig. 2B) was free in solution as
compared with 35% of compound U1 (average of three
samples). Adding sulfite to the reaction mixture eliminated compound
U1 completely and stimulated the formation of compound U2 (Fig. 2C). The relative amounts of compounds
U1 and U2 were variable in assays to which no
sulfite was added, depending on the SQD1 protein preparation. This
effect may have been caused by varying amounts of contaminating
compounds carried over from the E. coli extract in SQD1
preparations, a hypothesis that was not further investigated.
Compound U2 Has UDP-SQ-like Properties--
Until this
time, it has not been possible to identify compound U1.
Because of the lack of suitable standards, a de novo
structural elucidation would have been required for which we could not
obtain sufficient amounts of material. However, compound U2
co-chromatographed in the HPLC system with authentic UDP-SQ isolated
from the sqdD mutant of R. sphaeroides (Fig. 2,
C and D), indicating that this compound may be
the proposed intermediate of sulfolipid biosynthesis, UDP-SQ. To obtain
corroborating evidence, labeled compound U2 purified by
HPLC was analyzed by TLC together with extracts from [35S]sulfate-labeled R. sphaeroides wild type
and sqdD mutant cells. The latter are known to accumulate
UDP-SQ (11). Compound U2 co-chromatographed with UDP-SQ
also in this system (data not shown).
The sulfolipid synthase of spinach chloroplast envelopes is highly
discriminatory toward UDP-SQ (9, 10). We took advantage of the
substrate specificity of this enzyme and incubated compound U2 with spinach chloroplast membranes and observed the
formation of a 35S-labeled compound co-chromatographing
with sulfolipid (Fig. 3). Taken together,
these three independent lines of evidence identified compound
U2 as UDP-SQ.
Confirmation of Sulfite as the Sulfur Donor--
Thus far, the
greatest mystery in the elucidation of the biosynthetic pathway for
sulfolipid biosynthesis has been the nature of the sulfur donor for the
formation of UDP-SQ. The establishment of the SQD1 in vitro
assay described above gave us the opportunity to directly address this
problem. It seemed most likely that a metabolite or its derivative of
the sulfur assimilation pathway in bacteria and plants (sulfate, APS,
PAPS, sulfite, thiosulfate, sulfide, or sulfoglutathione) would provide
the sulfonic acid group in the formation of UDP-SQ. We therefore tested
these compounds unlabeled at concentrations ranging from 0.1 to 10 mM in the UDP-Glc-based SQD1 assay to determine whether
they could stimulate the formation of UDP-SQ. The addition of sulfate,
APS, and PAPS had no affect on the absolute amounts or ratios of the
reaction products. The addition of 0.1 mM thiosulfate,
sulfide, and sulfoglutathione resulted in a decrease in the relative
amount of compound U1 and an increase in the amount of
compound U2 as shown for sulfite in Fig. 1C. We
assumed that in all three instances, this effect was due to sulfite,
which was either produced by chemical reaction from thiosulfate,
sulfide, or sulfoglutathione in aqueous solution or was already present
in the respective compound preparations as contaminant.
To corroborate this hypothesis, it was necessary to directly test the
incorporation of labeled sulfite into UDP-SQ. Because labeled sulfite
was not commercially available and because it is fairly reactive in
solution (28), we decided to synthesize sulfite directly in the assay
mixture from [35S]APS and dithiothreitol as reductant
using recombinant APS reductase 1 from A. thaliana (APR1)
(25). The second SQD1 substrate, UDP-Glc, was provided unlabeled.
Incubating APS, dithiothreitol, UDP-Glc, and APR1 alone followed by
HPLC analysis of the reaction products resulted in the conversion of
APS to sulfite (Fig. 4, A and
B). When SQD1 was present in the APR1 reaction mixture,
sulfite was converted to compound U2 previously identified
as UDP-SQ (Fig. 4, C and D). The formation of
UDP-SQ from labeled APS in this APR1/SQD1 coupled assay was further
confirmed using the spinach sulfolipid synthase assay described above
(data not shown). Incubating labeled APS with SQD1 alone did not lead
to the formation of labeled UDP-SQ (result not shown, but essentially
indistinguishable from Fig. 4A).
A T145A Mutant of SQD1 with Strongly Decreased
Activity--
Sulfite had been previously suggested as a substrate for
sulfolipid synthesis using extracts of C. reinhardtii (22).
However, sulfite incorporation was not saturable as expected for
enzyme-catalyzed reactions in this system. To rule out a nonenzymatic
reaction of sulfite as the cause for the observed UDP-SQ formation and to demonstrate directly that SQD1 activity is required in the in
vitro assay system described above, we constructed a point mutant
of SQD1 (T145A) by exchanging threonine 145 with alanine (for gel, see
Fig. 1C). From the crystal structure of the
SQD1·UDP-Glc complex, it was obvious that threonine 145 coordinates a water along with the C-4 and C-6 hydroxyl groups of the
glucose moiety of UDP-Glc in a high energy conformation (16).
Therefore, it was predicted that threonine 145 plays a critical role
for catalytic activity. Indeed, when the T145A mutant was incubated for
40 min in the presence of sulfite and labeled UDP-Glc, no product was formed in comparison with the wild type reaction (Fig.
5, A and B). Only
after 46 h of incubation, a very small product peak was visible in
the mutant sample (Fig. 5C). This result suggested that the
activity of the mutant enzyme is reduced by several orders or
magnitude, thereby confirming that SQD1 enzymatic activity is essential
for the observed conversion of UDP-Glc and sulfite to UDP-SQ.
Characterization of SQD1 Activity--
Basic enzymatic properties
of SQD1 were determined using the standard assay as described under
"Experimental Procedures." Enzyme activity was linear from 5 to 50 µg of SQD1 protein as tested (Fig.
6A). The reaction was also
linear with respect to the assay time of up to 60 min (data not shown).
The optimal pH for activity was between 7.5 and 9.5 (Fig.
6B). Subsequently, all standard assays were performed at pH
7.5 with 10 µg of protein for 40 min. To determine the kinetic
constants for UDP-Glc, increasing amounts of this substrate were added
at a concentration of 100 µM sulfite (Fig.
6C). The reaction was saturable, and the Michaelis-Menten constant, Km, for UDP-Glc was estimated to be 150 µM, the specific activity 2.6 nmol of UDP-SQ
min Unlike sulfonic acids such as taurine, which represent oxidation
products of sulfur amino acids in animals, the sulfolipid head group
donor UDP-SQ (11) is synthesized de novo in bacteria and
plants. Studying the recombinant protein SQD1 of A. thaliana, we could observe the enzyme-catalyzed formation of
UDP-SQ in vitro. Although no direct structural elucidation
of the reaction product was feasible, three independent lines of
indirect evidence confirmed the identity of the product as UDP-SQ:
first, co-chromatography with authentic UDP-SQ by HPLC; second,
co-chromatography by TLC; and third, conversion of the product by
spinach SQDG synthase to sulfolipid. The formation of UDP-SQ was
dependent on the presence of UDP-Glc and sulfite. Based on this result,
we propose a tentative model of sulfolipid biosynthesis with UDP-Glc
and sulfite as the precursors as shown in Fig.
7.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-quinovosyl diacylglycerol
(SQDG)1 is a unique
nonphosphorous lipid found in the photosynthetic membranes of plants
and bacteria (1, 2). The head group of SQDG is sulfoquinovose, an
anionic sulfonic acid derivative of glucose (6-deoxy-6-sulfoglucose).
Therefore, SQDG contributes a negative charge to the thylakoid membrane
along with the other major anionic thylakoid lipid
phosphatidylglycerol. Sulfolipid-deficient bacterial mutants are
impaired in growth following phosphate deprivation (3, 4). Based on
this result and other evidence, it was proposed that SQDG is essential
to maintain a balance of thylakoid membrane charge by
substituting for phosphatidylglycerol under phosphate limiting
conditions (1).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Basic activity
assays were carried out at 37 °C in a buffer containing 10 µg of
SQD1, 100 µM Na2SO3, 500 µM UDP-[14C(U)]glucose (89 Bq
nmol
1), and 50 mM Tris (pH 7.5)
in a total volume of 100 µl for 40 min unless otherwise stated. For
some experiments, as indicated, we increased the UDP-Glc concentration
to 1.6 mM to fully saturate (10 × Km) the enzyme with this substrate. The coupled APS
reductase/SQD1 assay contained 50 mM Tris, pH 8.5, 10 mM dithiothreitol, 25 µM
[35S]APS (500 Bq nmol
1), 250 mM Na2SO4, 1 mM EDTA,
500 µM UDP-Glc, 66 µg of SQD1, and 12 µg of APR1 from
A. thaliana (25). This reaction was incubated at 30 °C
for 10 min. In all cases, samples were heat-denatured for 5 min at
95 °C, centrifuged at 10,000 × g for 5 min, and
analyzed by HPLC (Waters Corp., Milford, MA) employing a Beckman
(Fullerton, CA) Ultrasphere ODS column (4.6 mm × 25 cm; particle
size 5 µm) kept constantly at 42 °C. Substrates and products were
separated by applying a linear gradient of 30 mM
KH2PO4, 2 mM tetrabutylammonium hydroxide (Fisher), adjusted to pH 6.0 with KOH, to HPLC grade acetonitrile (EM Science, Gibbstown, NJ) with a flow rate of 1 ml/min
over 45 min. The column was allowed to re-equilibrate for 17 min
(acetonitrile to phosphate buffer) after each run. Labeled compounds
were detected using a
-Ram model 2 Flow Through Monitor (INUS
Systems, Tampa, FL).
1) as
substrate. The reactions were incubated at room temperature for 1 h. Assays were stopped by the addition of 100 µl of
chloroform/methanol/formic acid (1:1:0.1, v/v/v) and 50 µl of 1 M KOH, 0.2 M phosphoric acid. Samples were
mixed and centrifuged for 3 min at 10,000 × g. Lipids in the lower chloroform phase were analyzed by thin layer
chromatography on ammonium sulfate-impregnated plates by the method of
Benning and Somerville (6) with the substitution of toluene for benzene in the mobile phase. Autoradiography was used to visualize labeled sulfolipid.
RESULTS
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DISCUSSION
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Fig. 1.
Purification of SQD1. Shown are SDS-PAGE
analysis of crude E. coli cell culture extract expressing
SQD1 protein (A) and nickel column purification of SQD1
(B) and T145A mutant (C) (4 µg of each).
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Fig. 2.
Conversion of UDP-Glc by SQD1. The
chromatographic analysis of 14C-labeled substrate and
reaction products by HPLC is shown (A-C). Shown are UDP-Glc
without SQD1 protein (A), UDP-Glc and SQD1 protein
(B), UDP-Glc, SQD1 protein, and sulfite (C), and
authentic 35S-labeled UDP-SQ isolated from the
sqdD mutant of R. sphaeroides (D).
U1 and U2 are products as described under
"Results."
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Fig. 3.
Incubation of spinach chloroplast membranes
with reaction product U2. Assay of sulfolipid synthase
associated with thylakoid membranes, which specifically converts UDP-SQ
and diacylglycerol to SQDG. A, thin layer chromatography of
lipids following the incubation of spinach thylakoid membranes with
labeled reaction product U2 or, for control purposes,
14C-labeled UDP-Gal the substrate for galactolipid
biosynthesis. Lipids were visualized by autoradiography. B,
iodine staining of the U2 lane. DGDG,
digalactosyldiacylglycerol; MGDG,
monogalactosyldiacylglycerol; PC, phosphatidylcholine;
PG, phosphatidylglycerol.
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Fig. 4.
Coupled APS reductase/SQD1 assay. HPLC
chromatograms of reaction products and standards are shown.
A, 35S-labeled substrate APS without enzymes;
B, 35S-labeled reaction products following the
incubation with APS reductase alone or in the presence of APS reductase
and SQD1 (C). D, 14C-labeled UDP-SQ
(U2) from the standard SQD1 assay.
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Fig. 5.
Comparison of SQD1 wild type and T145A
mutant. HPLC chromatograms of reaction products are shown.
A, standard 40-min assay in the presence of SQD1 wild-type
enzyme. B, incubation with mutant enzyme for 40 min and
46 h (C).
1 mg
1 protein,
and the turnover number, kcat, 0.1 min
1. To examine the specificity of the
enzyme, we added equal amounts of ADP-Glc and UDP-Glc (500 µM each) with UDP-Glc as the labeled tracer. However, no
inhibition of the reaction by ADP-Glc was observed (data not shown).
Because SQD1 normally contains NAD+ in its binding site,
this nucleotide was added to the reaction, but it did not affect
product formation (data not shown). Keeping the UDP-Glc concentration
at 1.6 mM and varying the concentration of sulfite
(Fig. 6D), the reaction was saturable, with a
Vmax similar to that observed for UDP-Glc.
However, the Km for sulfite was ~10
µM, an order of magnitude lower as compared with UDP-Glc.
Increasing the sulfite concentration beyond 100 µM
inhibited the reaction (Table I). This
effect was specific to sulfite, because other salts as shown in Table I
did not inhibit the reaction.
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Fig. 6.
Exploring different assay conditions for
SQD1. A, the addition of varying amounts of SQD1
protein. B, dependence on pH. MES buffer was used for pH
values of 6.0, 6.5, and 7.0, Tris buffer for pH 7.5-9.5, and CAPS for
pH 10. C, substrate saturation for UDP-Glc at 100 µM sulfite. D, substrate saturation for
sulfite at 1.6 mM UDP-Glc. 13 µg of SQD1 protein was used
for analysis in B-D. Error bars, S.D.
for five samples.
SQD1 assay with excess sulfite and added salt
DISCUSSION
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ABSTRACT
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DISCUSSION
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Fig. 7.
Proposed model for sulfolipid biosynthesis in
plant chloroplasts. Enzymes involved are SQD1, ATP
sulfotransferase (ATS), APS reductase (APR), and
SQDG synthase. UDP-Glc is proposed to be imported from the cytosol as
indicated. DAG, diacylglycerol.
UDP-Glc as Substrate-- Sulfolipid biosynthesis is a function of chloroplasts (18-20), and SQD1 has previously been shown to be imported into the plastid (15). This poses a theoretical problem, because it is unclear whether the precursor UDP-Glc is actually available in the plastid, and, at least, one would have to assume that its concentration is very low compared with ADP-Glc (29). However, ADP-Glc, which is involved in photosynthetic starch biosynthesis in plastids, was not a substrate for the reaction. Furthermore, UDP-Glc provided a perfect fit within the active site based on the crystal structure of SQD1 (16). Therefore, we postulate that UDP-Glc is present in the plastid in sufficient amounts to support sulfolipid biosynthesis. Whether UDP-Glc is imported from the cytosol as indicated in Fig. 7 or generated inside the plastid remains unclear.
Sulfite Is the Sulfur Donor-- Of all of the possible sulfur donors tested, none was more active than sulfite. Compounds that could spontaneously give rise to sulfite in aqueous solution, such as thiosulfate and sulfoglutathione, did stimulate the activity of the enzyme to the same extent as sulfite directly. It is still debated whether APS-reductase generates sulfite directly or first generates sulfoglutathione, which subsequently hydrolyzes to release sulfite (30, 31). Therefore, the question arises of whether sulfoglutathione may be a precursor for UDP-SQ biosynthesis in vivo. Two arguments speak against this hypothesis: first, glutathione could not be modeled into the structure of SQD1 in a catalytically sensible way; second, sulfoglutathione was not more effective in stimulating UDP-SQ biosynthesis than sulfite itself. Therefore, we propose that sulfite derived from the APS reductase reaction is the precursor for UDP-SQ biosynthesis in vivo. The APS-reductase is part of the sulfur assimilation machinery in the chloroplast. At least three isoforms exist (32), and it seems possible that SQD1 directly and specifically interacts with one of these. Sulfite is a reactive and cytotoxic compound (e.g. Refs. 33-35), and substrate channeling between one of the APS reductase isoforms and SQD1 would allow sulfolipid biosynthesis without build-up of sulfite as an intermediate. The Km of ~10 µM for sulfite is relatively low in accordance with a high affinity of SQD1 for the substrate. Interestingly, concentrations above 100 µM sulfite inhibit the reaction. Whether this inhibition reflects a biologically meaningful regulatory process or a toxic effect on the protein due to reactive radicals derived from sulfite oxidation (28) requires further investigation.
The SQD1-catalyzed Formation of UDP-SQ Is Very Slow-- Contrary to experiments with extracts of C. reinhardtii (22), the formation of UDP-SQ from sulfite and UDP-Glc was saturable with increasing amounts of sulfite as would be expected for an enzyme-catalyzed reaction. The reaction depended on the presence of SQD1 enzyme, and evidence for the crucial role of SQD1 in the formation was derived from the T145A mutant of SQD1, which was virtually inactive while retaining a native structure.2 Although clearly measurable, the reaction of the wild-type protein is already very slow, with 0.1 turnovers/min. It seems unlikely that the activity of SQD1 is this low in vivo because it would presumably not suffice to produce enough sulfolipid during rapid leaf growth. Assuming that UDP-Glc and sulfite are the correct substrates, at least three explanations can be found for the low in vitro activity of SQD1. First, the recombinant enzyme has been truncated at the N terminus to remove the predicted transit peptide (15). The prediction of the cleavage site may be incorrect, and the truncation may have affected activity. However, the bacterial SQDB proteins are similar in size as compared with the recombinant SQD1 protein and seem to work properly in vivo. Second, an allosteric factor is missing that would normally activate the enzyme in vivo. Third, SQD1 is part of a larger protein complex and requires, thus, direct and proper contact with an APS reductase and possibly other enzymes. At this time, we cannot distinguish between these possibilities. Another unusual feature of SQD1 activity is that it shows a broad pH optimum, between 7.5 and 9.5. One possibility for the high activity close to pH 7.5 is that it arises from the change in protonation state of Tyr182 and/or His183. The amino acid Tyr182 is thought to initiate catalysis by abstracting a proton from the 4'-hydroxyl group of UDP-Glc. A homologous residue in the structurally related enzyme UDP-galactose 4'-epimerase, Tyr149, has an estimated pKa of 6.08 (36). If Tyr182 of SQD1 had a similar pKa, SQD1 would lose activity as the pH decreased. Moreover, His 183 is considered to be the general base in the dehydration step that requires the removal of the C-5' proton from glucose (16). Unless perturbed by the local environment, His183 should have a pKa of ~7. The origin of the high activity close to pH 9.5 remains an enigma.
In summary, we have shown that recombinant SQD1 of A. thaliana catalyzes the formation of UDP-SQ, the sulfolipid head
group donor and one of the few biological sulfonic acids, in
vitro from UDP-Glc and sulfite. The reaction showed all features
expected for an enzymatic reaction. However, the turnover rate was very low and further analysis will be required to demonstrate that SQD1
catalyzes the proposed reaction also in vivo.
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ACKNOWLEDGEMENTS |
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We thank J. F. Leykam for helpful discussions during the development of the HPLC assay, Liqun Mao for a contribution during a laboratory rotation, and Bart Leonard for technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Science Foundation Grant MCB-9807993 (to C. B.) and a grant from the MSU Research Excellence Fund Center for Protein Structure, Function, and Design.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 and reprint requests should be addressed. Tel.: 517-355-1609; Fax: 517-353-9334; E-mail: benning@pilot.msu.edu.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M008200200
2 M. J. Theisen, S. Sanda, C. Benning, and R. M. Garavito, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
SQDG, sulfolipid
6-sulfo--D-quinovosyl diacylglycerol;
UDP-SQ, UDP-sulfoquinovose;
UDP-Glc, UDP-glucose;
UDP-Gal, UDP-galactose;
HPLC, high pressure liquid chromatography;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
CAPS, 3-(cyclohexylamino)propanesulfonic acid;
MES, 4-morpholineethanesulfonic acid.
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1. | Benning, C. (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 53-75[CrossRef] |
2. | Harwood, J. L. (1980) in The Biosynthesis of Plants (Stumpf, P. K., ed), Vol. 4 , pp. 301-320, Academic Press, Inc., New York |
3. | Benning, C., Beatty, J. T., Prince, R. C., and Somerville, C. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1561-1565[Abstract] |
4. |
Güler, S.,
Seeliger, A.,
Härtel, H.,
Renger, G.,
and Benning, C.
(1996)
J. Biol. Chem.
271,
7501-7507 |
5. | Benning, C., and Somerville, C. R. (1992) J. Bacteriol. 174, 6479-6487[Abstract] |
6. | Benning, C., and Somerville, C. R. (1992) J. Bacteriol. 174, 2352-2360[Abstract] |
7. | Rossak, M., Schäfer, A., Xu, N., Gage, D. A., and Benning, C. (1997) Arch. Biochem. Biophys. 340, 219-230[CrossRef][Medline] [Order article via Infotrieve] |
8. | Benson, A. A. (1963) Adv. Lipid Res. 1, 387-394 |
9. | Heinz, E., Schmidt, H., Hoch, M., Jung, K. H., Binder, H., and Schmidt, R. R. (1989) Eur. J. Biochem. 184, 445-453[Abstract] |
10. | Seifert, U., and Heinz, E. (1992) Bot. Acta 105, 197-205 |
11. |
Rossak, M.,
Tietje, C.,
Heinz, E.,
and Benning, C.
(1995)
J. Biol. Chem.
270,
25792-25797 |
12. | Tietje, C., and Heinz, E. (1998) Planta 206, 72-78[CrossRef] |
13. | Weissenmayer, B., Geiger, O., and Benning, C. (2000) Mol. Plant Microbe Interact. 13, 666-672[Medline] [Order article via Infotrieve] |
14. | Pugh, C. E., Roy, A. B., Hawkes, T., and Harwood, J. L. (1995) Biochem. J. 309, 513-519[Medline] [Order article via Infotrieve] |
15. |
Essigmann, B.,
Güler, S.,
Narang, R. A.,
Linke, D.,
and Benning, C.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
1950-1955 |
16. |
Mulichak, A. M.,
Theisen, M. J.,
Essigmann, B.,
Benning, C.,
and Garavito, R. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13097-13102 |
17. | Essigmann, B., Hespenheide, B. M., Kuhn, L. A., and Benning, C. (1999) Arch. Biochem. Biophys. 369, 30-41[CrossRef][Medline] [Order article via Infotrieve] |
18. | Haas, R., Siebertz, H. P., Wrage, K., and Heinz, E. (1980) Planta 148, 238-244 |
19. | Joyard, J., Blee, E., and Douce, R. (1986) Biochim. Biophys. Acta 879, 78-87 |
20. | Kleppinger-Sparace, K. F., Mudd, B., and Bishop, D. G. (1985) Arch. Biochem. Biophys. 240, 859-865[Medline] [Order article via Infotrieve] |
21. | Kleppinger-Sparace, K. F., and Mudd, J. B. (1990) Plant Physiol. 93, 256-263 |
22. | Hoppe, W., and Schwenn, J. D. (1981) Z. Naturforsch. 36c, 820-826 |
23. | Eriksson, B., and Rundfelt, M. (1968) Acta Chem. Scand. 22, 562-570[Medline] [Order article via Infotrieve] |
24. | Eriksson, B., and Eriksson, S. A. (1967) Acta Chem. Scand. 21, 1304-1312[Medline] [Order article via Infotrieve] |
25. |
Bick, J. A.,
Aslund, F.,
Chen, Y.,
and Leustek, T.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8404-8409 |
26. | Lichtenthaler, H. K. (1987) Methods Enzymol. 148, 350-382 |
27. | Ito, W., Ishiguro, H., and Kurosawa, Y. (1991) Gene (Amst.) 102, 67-70[CrossRef][Medline] [Order article via Infotrieve] |
28. | Hayon, E., Treinin, A., and Wilf, J. (1972) J. Amer. Chem. Soc. 94, 47-57 |
29. |
Bligny, R.,
Gardestrom, P.,
Roby, C.,
and Douce, R.
(1990)
J. Biol. Chem.
265,
1319-1326 |
30. | Hell, R. (1997) Planta 202, 138-148[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Leustek, T.,
and Saito, K.
(1999)
Plant Physiol.
120,
637-643 |
32. | Bick, J. A., and Leustek, T. (1998) Curr. Opin. Plant Biol. 1, 240-244[CrossRef][Medline] [Order article via Infotrieve] |
33. | Tuazon, P. T., and Johnson, S. L. (1977) Biochemistry 16, 1183-1185[Medline] [Order article via Infotrieve] |
34. | Shimazaki, K., Nakamachi, K., Kondo, N., and Sugahara, K. (1984) Plant Cell Physiol. 25, 337-341 |
35. | Lizada, M. C. C., and Yang, S. F. (1981) Lipids 16, 189-194 |
36. | Liu, Y., Thoden, J. B., Kim, J., Berger, E., Gulick, A. M., Ruzicka, F. J., Holden, H. M., and Frey, P. A. (1997) Biochemistry 36, 10675-10684[CrossRef][Medline] [Order article via Infotrieve] |