(Received for publication, August 15, 1995)
From the
Biosynthesis of the activated sulfate donor, adenosine
3`-phosphate 5`-phosphosulfate, involves the sequential action of two
enzyme activities: ATP sulfurylase, which catalyzes the formation of
adenosine 5`-phosphosulfate (APS) from ATP and free sulfate, and APS
kinase, which subsequently phosphorylates APS to produce adenosine
3`-phosphate 5`-phosphosulfate. Oligonucleotide primers were derived
from a human infant brain-expressed sequence tag putatively encoding a
portion of APS kinase. Using these primers, reverse
transcriptase-polymerase chain reaction was performed on mRNA from
neonatal normal mice resulting in amplification of a 127-bp DNA
fragment. This fragment was subsequently used to screen a mouse brain
gt11 cDNA library, yielding a 2.2-kb clone. Primers were designed
from the 5`-end of the 2.2-kb clone, and 5`-rapid amplification of cDNA
ends was used to obtain the translation start site. Sequence from the
overlapping clones was assembled into a 2475-bp composite sequence,
which contains a single open reading frame that translates into a
624-deduced amino acid sequence. Northern blots of total RNA from
neonatal mice yielded a single message species at approximately 3.3 kb.
Southern blot of genomic DNA digested with several restriction enzymes
suggested the gene is present as a single copy. Comparison against
sequence data bases suggested the composite sequence was a fused
sulfurylase-kinase product, since the deduced amino acid sequence
showed extensive homology to known separate sequences of both ATP
sulfurylase and APS kinase from several sources. The first 199 amino
acids corresponded to APS kinase sequence, followed by 37 distinct
amino acids, which did not match any known sequence, followed by 388
amino acids that are highly homologous to known ATP sulfurylase
sequences. Finally, recombinant enzyme expressed in COS-1 cells
exhibited both ATP sulfurylase and APS kinase activity.
Sulfate activation involves the transfer of a sulfate group to
ATP by ATP sulfurylase (ATP sulfate adenylyltransferase, EC 2.7.7.4) to
yield adenosine 5`-phosphosulfate (APS) ()and pyrophosphate.
Subsequently, APS kinase (ATP adenosine-5`-phosphosulfate
3`-phosphotransferase, EC 2.7.1.25) transfers a phosphate group from
ATP to APS to yield ADP and adenosine 3`-phosphate 5`-phosphosulfate
(PAPS). Since the equilibrium for the ATP sulfurylase reaction is
rather unfavorable (K
=
10
) in the physiologic direction, APS kinase plays
an important role by continually removing APS, thus driving the overall
sulfate activation pathway in the forward direction. Moreover, APS
itself is structurally unstable and is subject to spontaneous
degradation under physiologic conditions. PAPS is the sole source of
sulfate for sulfate esters in mammals, and APS appears to be only an
intermediate in the sulfate-activating pathway.
The purification of ATP sulfurylase and APS kinase from a single species has been reported for the fungus Penicillium chrysogenum(1, 2) and Escherichia coli(3, 4) . ATP-sulfurylase has been purified from a number of lower organisms (5, 6, 7, 8) , usually varies between 42 and 67 kDa, and may form oligomers. In addition to fungi and bacteria, APS-kinase has been isolated from Chlamydomonas reinhardii(9) ; most kinases of these lower organisms are small (21-44 kDa) and often form dimers. Although ATP sulfurylase has been previously cloned in Saccharomyces cerevisiae(10) , E. coli(11) , Arabidopsis thaliana(12) , Rhizobium meliloti(13) , P. chrysogenum(14) , and Riftia pachyptila(15) , and although APS kinase has been cloned in E. coli(16) and R. meliloti(17) , neither enzyme has been cloned from a mammalian source.
We have had a long-standing interest in the sulfate-activation pathway since characterizing the unique defect in the brachymorphic mouse, which affects both ATP sulfurylase and APS kinase(18, 19) . Attempts to purify these two enzyme activities from rat chondrosarcoma showed that they copurifed over 2000-fold, suggesting that the activities are inseparable(20) . Recent studies have demonstrated that rat chondrosarcoma ATP sulfurylase and APS kinase reside on a single bifunctional enzyme(21) , which uses a channeling mechanism to efficiently synthesize PAPS(22) . Most recently, we demonstrated that the defect in the brachymorphic mouse results from a mutation that primarily alters the function of the novel coupling mechanism between the two active sites(23) . Therefore, we have directed our efforts toward cloning and sequencing this unique enzyme to further understand the channeling function and eventually elucidate the brachymorphic defect. Here, we report the isolation of a 2475-bp cDNA from Mus musculus, which yields a 624-deduced amino acid sequence encoding both ATP sulfurylase and APS kinase activities.
Transient transfections of
COS-1 cells were performed by the calcium phosphate procedure as
described(24) . Briefly, the cells were transfected with 20
µg of DNA and incubated at 37 °C for 12 h in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum. After 12 h,
the growth medium was changed, and the cells were incubated at 37
°C for an additional 48 h. The cells were then washed twice with
phosphate-buffered saline and scraped with a rubber policeman into
sucrose-phosphate buffer (50 mM
NaHPO
-K
HPO
, pH 7.8,
0.25 M sucrose, 0.25 M KCl, 1 mM EDTA). The
cells were sonicated briefly and centrifuged at 10,000 rpm, and enzyme
assays were performed on supernatant fractions.
The standard kinase assay (29) contained 10
nM [S]APS, 250 µM ATP (pH
7), 5 mM MgCl
, 10 mM ammonium sulfate,
and 12 µl of enzyme and was brought to 25 µl with buffer A (25
mM NaH
PO
-K
HPO
,
pH 7.8, 1 mM dithiothreitol, 1 mM EDTA, and 10%
glycerol).
The coupled assay has been recently
developed(22) . The standard 25-µl reaction mixture
contained 0.4 mM
[S]H
SO
, 10 mM ATP, 20 mM MgCl
, 22 mM Tris-HCl (pH
8.0), and 10 µl of enzyme preparation.
Sequence
Analysis-Sequence data base searches were performed using
BLAST programs (30) on the data bases maintained by the
National Center for Biotechnology Information at the National Library
of Medicine. Nucleotide sequence from the mouse clones was assembled,
and the deduced amino acid sequence was generated and analyzed using
the computer programs SEQED, LINEUP, TRANSLATE, PEPTIDESORT, GAP,
COMPARE, DOTPLOT, and PILEUP of the Wisconsin Package. ()
This probe
was then used to screen approximately 500,000 recombinants of an
18-day-old mouse brain gt11 cDNA library. Recombinants were
replica-plated onto nitrocellulose filters and probed with the 127-bp
fragment. A single 2.2-kb clone (SK 9-1) was identified and
subsequently plaque-purified, subcloned and sequenced, and was found to
lack a probable initiation codon. Therefore, two new primers were
designed from the SK 9-1 5`-end, and 5`-rapid amplification of cDNA
ends was used to obtain a 300-bp cDNA, which was subcloned and
sequenced and found to contain the translation initiation codon.
Multiple sequence determinations were made for all primers used, and
uncertainties in the gel patterns were resolved by sequencing with a
different polymerase, sequencing the complementary strand, or both.
Sequence from the overlapping clones was assembled into a 2475-bp composite sequence, which contains a single open reading frame that translated into a 624-deduced amino acid sequence having a calculated molecular mass of 70.7 kDa, the longest obtainable from our data (Fig. 1). This composite contains a region that correlates closely to an ATP-GTP binding motif (P-loop) (32, 33) from amino acid residue 59 to 65, flanked by cysteine residues at position 53 and 78 (and 83), the FISP sequence at residues 131-134(3) , and a PAPS-dependent enzyme motif from residue 175 to 186(34, 35) . Toward the C-terminal end, a sequence commensurate with the recently described PP-motif found in ATP sulfurylases and PAPS reductases (36) is also present (residues 521-525).
Figure 1: cDNA and deduced amino acid sequences of mouse brain ATP sulfurylase APS kinase. The composite cDNA sequence of 2475 nucleotides is shown with the deduced amino acid sequence of 624 residues beginning at nucleotide 34. The first 1-199 residues corresponding to know APS kinase, and residues 237-624 corresponding to known APS sulfurylase sequences are shaded. The P-loop motif (residues 59-65) and the PAPS-dependent enzyme motif (residues 175-186) are boxed, as are two highly conserved sequences (residues 411-431 and 499-523) found in other ATP sulfurylases. Invariant Cys flanking the P-loop (residues 53, 78, and 83) and several invariant Lys, His, and Arg found in other ATP sulfurylases are circled.
The Wisconsin Package program COMPARE was used for pairwise comparisons between the putative sulfurylase-kinase sequence and each of the known individual sulfurylase and kinase sequences from several sources, both to localize the kinase and sulfurylase domains and to check for the presence of repeating elements or internal rearrangements. The results were displayed with the program DOTPLOT, and outcomes of pairwise comparisons with Arabidopsis and Saccharomyces sequences are shown in Fig. 2. Each plotted point represents a register of alignment and window location at which 15 of 30 residues in the window matched; identical or very similar colinear sequences result in a single diagonal line with a slope of 1. Repeats occurring in both sequences produce pairs of shorter diagonals paralleling the main register line, and a gap in one sequence of a pair is seen as a break or displacement of the main line. The APS-kinase comparisons (Fig. 2, A and C) reveal the greatest amount of colinear similarity across species, as represented by the dominant diagonal line. The ATP sulfurylase comparisons show more variability among species, with greater similarity to Arabidopsis than to Saccharomyces (Fig. 2, B and D).
Figure 2: Comparison of A. thaliana, S. cerevisiae, and M. musculus ATP sulfurylase and APS kinase deduced amino acid sequences. Dot matrix comparison of the deduced amino acid sequence of mouse ATP sulfurylase-APS kinase against A. thaliana APS kinase (A), A.thaliana ATP sulfurylase (B), S. cerevisiae APS-kinase (C), and S. cerevisiae ATP sulfurylase (D) sequences obtained using the Wisconsin package programs COMPARE (window = 30, stringency = 15) and DOTPLOT. The A. thaliana APS kinase, A. thaliana ATP sulfurylase, S. cerevisiae APS kinase, and S. cerevisiae ATP sulfurylase sequences are translations of the GenBank DNA sequences with accession numbers U05238, U05218, S55315, and X60157, respectively.
A multiple
alignment of the mouse sequence and representative APS kinases and ATP
sulfurylases was done to examine the detail of the molecules'
similarities (Fig. 3). Based on those alignments, we postulate
that the first 199 amino acids (nucleotides 34-630) correspond to
APS kinase activity. This region is followed by a 37-amino acid stretch
(nucleotides 631-742), which is not similar to either ATP
sulfurylase or APS kinase. Subsequent amino acids from 237 to 624
(nucleotides 743-1905) are highly homologous to sequences with known
ATP sulfurylase activity. As mentioned, the region that correlates
closely to an ATP-GTP binding motif (P-loop) and the FISP sequence are
identified only in the putative APS-kinase sequence and are also
flanked by several highly conserved cysteine residues, as previously
reported in Arabidopsis(35) . There are also two
invariant lysines and seven invariant arginines, often as part of
homologous sequences, across the APS kinases that have been cloned and
sequenced. As well, conserved regions containing invariant Arg and His
residues analogous to those found in previously cloned ATP sulfurylases
and proposed to be involved in MgATP and SO binding (14) are present in the APS sulfurylase portion
of the new mouse sequence at residues 411-431 with invariant
Arg-421, His-425, and His-428 and 499-523 with invariant His-506,
Arg-510, and Arg-522. These specific features, as well as overall
homology to known ATP sulfurylase and APS kinase sequences from
multiple species, suggest that this new deduced sequence represents a
fused sulfurylase-kinase product.
Figure 3: Alignment of various ATP sulfurylase and APS kinase sequences from several species. A, schematic diagram of APS-kinase and ATP-sulfurylase domains identified by protein analysis. P-loop motif (*), PAPS-dependent enzyme motif (**), and PP-motif (***) are indicated. B, the peptide sequences aligned to the mouse sulfurylase-kinase sequence were obtained by translation of the following GenBank entries with data base accession numbers of the sequences in parentheses: Rmel-nodQ (M68858), Eco-cysC (M74586), Scer-kin(555315), Athal-kin (U05238), HumEST-kin (T09181), Stub-sulf X79053), Athal-sulf (U05218), Pchry-sulf (U07353), and Scer-sulf (X60157). Kinase and sulfurylase sequences were separately aligned to the mouse sulfurylase-kinase (shaded) sequence using the program PILEUP. The two groups of sequences were then merged with the program LINEUP using the PRETTY output option. Invariant residues are boxed; proposed functional domains are designated (*) and defined in the text.
Figure 4:
Northern blot analysis of ATP sulfurylase
APS kinase RNA. 30 µg of total RNA from neonatal mouse brain was
loaded on a 1% agarose gel. The blot was hybridized with a P-labeled 2.2-kb probe to yield a single message species
at approximately 3.3 kb (lane 1). As a control, the same blot
was stripped and hybridized with a mouse glyceraldehyde-3-phosphate
dehydrogenase cDNA probe (lane 2). The location of RNA size
markers (in kb) is shown on the left. Autoradiograph was
exposed for 5 days; prolonged exposure did not reveal any additional
message species.
Figure 5: Southern blot of mouse genomic DNA. Two different isolations of mouse genomic DNAs were digested with the indicated restriction endonucleases. 15 µg of DNA were loaded in each lane and electrophoresed on 1% agarose gel. The probe was the 127-bp fragment used to screen the cDNA library. Size markers are indicated on the right.
Figure 6: Transient expression analysis of the ATP sulfurylase APS kinase recombinant enzyme. Constructs in the pSVL vector of a version of sulfurylase-kinase (from residues 20 to 624) in the correct genomic orientation (bar 1), in the reverse orientation (bar 2), or empty vector (bar 3) were used to transfected COS-1 cells using a calcium phosphate procedure as described under ``Experimental Procedures.'' Specific activities of ATP-(reverse) sulfurylase (left panel), APS kinase (central panel), and overall ATP-sulfurylase-APS-kinase reaction were determined using the supernatant fractions of sonicated cells as described(21, 27, 29) . Bars 1 and 2 represent the average of six independent transfections. Base-line specific activities in non-transfected COS-1 cells are shown in bar 4 of each panel.
ATP sulfurylase and APS kinase are essential enzymes in sulfate activation. In lower organisms, these enzymes appear to be relatively small, contained on separate proteins, and supply PAPS mainly for cysteine biosynthesis. Three gene products are required for sulfate activation in E. coli(11) : CysD and CysN encode ATP sulfurylase and CysC encodes APS kinase. ATP sulfurylase is found as a heterodimer of 27- and 62-kDa subunits(4) , in which the 27-kDa peptide is the catalytic subunit (CysD), while CysN forms the 62-kDa GTP binding/hydrolyzing subunit(11) . Leyh and Suo (11) have studied the gene organization of the sulfate activation locus in E. coli and noted that the translational termination and initiation sequences of the CysD-CysN and CysN-CysC gene pairs overlap. The gene order is therefore ATP sulfurylase followed by APS kinase. Translational coupling in which the termination and initiation sequences of the CysD-CysN and CysN-CysC gene pairs overlap has been implicated in maintaining stoichiometry as well as enhancing translation efficiency (37, 38) .
Interestingly, Foster et al.(14) have demonstrated via cloning and sequencing that the C-terminal domain of P. chrysogenum ATP sulfurylase is similar to the nucleotide sequence of APS kinases from several organisms. Although this portion of the ATP sulfurylase does not confer APS kinase activity on the enzyme, Foster et al.(14) postulate that a large region of the ATP sulfurylase may have evolved from APS kinase and that, therefore, this region may contain the allosteric PAPS binding site. Second, Schwedock et al.(17) have recently shown that the nodulation gene nodP encodes an ATP sulfurylase, while the product of the nodQ gene has APS kinase activity in addition to its role in ATP sulfurylase in R. meliloti. The findings of fused domains for ATP sulfurylase and APS kinase in these two systems are both evolutionarily and mechanistically relevant to our findings of a mammalian bifunctional sulfurylase-kinase enzyme(21) , which also uses a channeling mechanism to efficiently synthesize PAPS(22) .
We have now isolated overlapping cDNA fragments
whose composite sequence is postulated to encode a fused sulfurylase-
kinase product based on the following criteria. The encoded protein
derives from a single reading frame and exhibits a molecular weight
commensurate with that previously obtained for the native bifunctional
enzyme(21) ; when compared to protein sequence data bases, the
deduced amino acid sequence shows extensive homology to separate
sequences of both ATP sulfurylases and APS kinases from several
sources; the expressed recombinant protein catalyzed the synthesis of
both APS (ATP sulfurylase activity) and PAPS (APS kinase activity) as
well as overall activity (i.e. synthesizing PAPS from ATP and
SO). Furthermore, the mouse
sulfurylase-kinase sequence contains highly conserved residues, which
are found in all ATP sulfurylases that have been sequenced, and are
postulated to be involved in MgATP and SO
binding(14) . There are also invariant Lys and Arg
residues, often a part of a homologous sequence, across all six APS
kinases that have been sequenced, including now the mouse
sulfurylase-kinase. In addition, the kinase domain exhibits a region
that correlates closely to a ATP-GTP binding motif (P-loop), as might
be expected for a phosphate binding protein (33) . The sequence
(GXXGXGK(TT)) is identical to the pattern in
thymidine kinase (39) and to the octapeptide signature
(GESGAGKT) in myosin heavy chain(40) . A PAPS-dependent enzyme
motif (KAXAGXXXXFTG) (34, 35) is
also present in the N-terminal APS-kinase portion. A sequence
resembling a portion of the recently proposed ATP pyrophosphatase PP
motif(36) , found in several ATP sulfurylases as well as PAPS
reductase, was also found in the mouse brain sulfurylase-kinase
sequence.
Although all these data strongly suggest that the fused
mammalian sulfurylase-kinase is related to similar activities
previously isolated and cloned from lower organisms, the gene
organization found in E. coli and P. chrysogenum differs significantly from the organization of the product we have
isolated. Our sequence shows strong homology in the N-terminal domain
to known APS kinases and strong homology in the C-terminal domain to
known ATP sulfurylases. This is the opposite orientation from the
fungal ATP sulfurylase, which has 75% of APS kinase at its C-terminal
sequence. In addition to resulting in interspecies structural
differences, this reverse orientation (as well as the fact that most of
the sulfurylases and kinases are clearly separate gene products) may
contribute to significant mechanistic differences. Rat chondrosarcoma
sulfurylase-kinase releases PP followed by APS, with
concomitant binding of the APS to the APS kinase activity, while P.
chrysogenum sulfurylase releases APS first followed by
PP
. The former specific order of product release and
substrate addition may result in a more efficient pathway via substrate
channeling of the APS intermediate, as we have demonstrated occurs in
the rat chondrosarcoma enzyme system(22) . In contrast, APS
bound to the ATP sulfurylase does not serve as a substrate for the APS
kinase of P. chrysogenum(39) , suggesting a different
mechanism pertains for the fungal ATP sulfurylase fused with a partial
APS kinase sequence.
The combined ATP sulfurylase-APS kinase described in this paper may represent an evolutionary trend toward a more efficient sulfate activation pathway in higher organisms. Although procaryotes can achieve coordinate expression of consecutive reactions through linkage of different polypeptides in a single operon, eucaryotic coordinate expression is more often realized by linking multiple functions in a single polypeptide. Bazan et al.(41) have proposed that certain bifunctional enzymes such as 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase represent the gene fusion of catalytic units. The newly cloned mouse brain bifunctional sulfurylase-kinase may have followed such a combinatorial process to maintain stoichiometry of the two activities and to achieve the maximal functional efficiency necessary for optimal PAPS production.
The cloning of sulfurylase-kinase will allow its role in the PAPS activation pathway to be studied further in higher organisms. Homology of the single open reading frame-generated sequence to known ATP sulfurylases and ATP kinases and demonstrable activity of the recombinant protein verify our earlier work that the sulfurylase-kinase in mammals is represented by a single bifunctional enzyme(21) . Further expression analysis of mutated forms will allow clarification of mechanistic and evolutionary structure-function relationships for this essential enzyme, as well as provide a library of phenotypic mutants for future pathophysiological studies and eventually a basis for elucidation of the molecular defect in the brachymorphic mouse.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34883[GenBank].