Identification and Functional Characterization of the Novel BM-motif in the Murine Phosphoadenosine Phosphosulfate (PAPS) Synthetase*

Bhawani Singh and Nancy B. SchwartzDagger

From the Departments of Chemistry, Biochemistry & Molecular Biology, and Pediatrics and the Committee of Developmental Biology, The University of Chicago, Chicago, Illinois 60637

Received for publication, July 5, 2002, and in revised form, October 22, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PAPS synthetase (SK) catalyzes the two sequential reactions of phosphoadenosine phosphosulfate (PAPS) synthesis. A functional motif in the kinase domain of mouse SK, designated the BM-motif (86 LDGDNhRxhh(N/S)(K/R)97), was defined in the course of identifying the brachymorphic (bm) defect. Sequence comparison and the secondary structure predicted for APS kinase suggest that the BM-motif consists of a DGD-turn sequence flanked by other conserved residues. Mutational analysis of the DGD-turn revealed that a flexible and neutral amino acid is preferred at residue 88, that negatively charged residues are strictly required at positions 87 and 89, and that the active site is rigid. The reduction in kinase activity for all DGD-turn mutants, except G88A, was much less severe than the reduction in overall activity, indicating that the BM-motif may also be playing a role in adenosine phosphosulfate (APS) channeling. Two switch mutations, LD86DL and DN89ND, designed to test the positional constraints of Asp87 and Asp89, exhibited complete loss of both kinase and overall activities, while LD86DL also exhibited a significant (60%) loss of reverse sulfurylase activity, suggesting that this peptide region is interacting with the sulfurylase domain as well as functioning in the kinase reaction. Other residues targeted for mutational analysis were the highly conserved flanking Asn90, Arg92, and Lys97. N90A resulted in a partial (30%) loss in kinase and overall activities, R92A exhibited total loss of kinase and overall activities, and K97A had no effect on any of the three activities. The complexity of the bifunctional SK in catalyzing the kinase reaction and channeling APS is illustrated by the strict requirements of this novel structural motif in the kinase active site.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sulfation is the second most common chemical modification of biomolecules, and in higher organisms all sulfation of carbohydrates, lipids, and protein substrates is mediated through the universal sulfate donor, phosphoadenosine phosphosulfate (PAPS)1 (1). The synthesis of PAPS is catalyzed by the bifunctional enzyme PAPS synthetase in higher organisms. PAPS synthetase consists of two activities, ATP sulfurylase, which catalyzes synthesis of adenosine phosphosulfate (APS) from ATP and SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and APS kinase, which phosphorylates APS in the presence of another molecule of ATP to form PAPS.
           <UP>ATP</UP>+<UP>SO</UP><SUP>−2</SUP><SUB>4</SUB> → <UP>APS</UP>+<UP>PP<SUB>i</SUB></UP> (Eq. 1)

<UP>APS</UP>+<UP>ATP</UP> → <UP>PAPS</UP>+<UP>ADP     </UP>
An animal model with reduced sulfation, the brachymorphic (bm) mouse, was initially identified by Sugahara and Schwartz (2) to possess a defective PAPS synthesis pathway (3, 4). Murine brachymorphism is a severe growth disorder, affecting the skeletal elements as well as certain physiological processes like blood clotting (5) and liver-mediated detoxification (6). Biochemical analysis showed that brachymorphic cartilage contains normal levels of glycosaminoglycans but has disaccharides that are significantly under-sulfated. The reduced incorporation of sulfate into brachymorphic cartilage is associated with limited PAPS availability largely due to a reduction in APS-kinase activity (2, 4).

The initial cloning of a bifunctional PAPS synthetase (SK1) did not identify the underlying etiology of the brachymorphic phenotype, thus prompting the subsequent elucidation of a second isoform (SK2) encoded by a separate gene (7). Sequence analysis of wild-type and bm SK2 cDNAs revealed that a G to A mutation in the bm allele changes a glycine to an arginine residue (G79R) in the APS-kinase domain of the bifunctional enzyme. Bacterially expressed recombinant normal SK2 protein catalyzes both the ATP sulfurylase and APS kinase reactions and synthesizes PAPS comparably to SK1. In contrast, recombinant SK2 protein containing the G79R mutation had no APS-kinase activity while maintaining its ATP sulfurylase activity. This study provided the first indication that the peptide region in the vicinity of residue 79 in SK2 may also be important for PAPS synthesis.

Amino acid sequence alignment of various monofunctional ATP sulfurylase and APS kinase enzymes from lower organisms and plants and bifunctional enzymes from animals revealed the presence of several highly conserved regions (8), e.g. the ATP binding (P)-loop (9), phosphoryl transfer (FISP) (10), phosphodiester-cleavage (11), and pyrophosphate binding (PP)-loop (11), all of which we have shown by mutational analysis to confer molecular binding or enzymatic activity to SK1. Similarly, sequence conservation around the bm mutation site (Gly79) is very high; however, no function had been established for this region. Based on the sequence alignment shown in Fig. 1, the proposed consensus sequence of the region around Gly79, called the BM-motif, is LDGDNhRxhh(N/S)(R/K), where h is a hydrophobic residue and x is a non-conserved residue. The available monofunctional fungal APS kinase structure (12) shows that the homologous region in the fungal enzyme consists of a beta -strand (residues 56-60) and an alpha -helix (residues 62-68), with Asp61 present between these two peptide structures. Although MacRae et al. were unable to crystallize the APS kinase (12) with bound ATP or APS, comparisons with substrate-bound structures of guanylate kinase (13) or the 6-phosphofructo-2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (14) suggest that Asp61 could be binding to the magnesium of MgATP. Thus, to explore the functional significance of the BM-motif in the bifunctional PAPS synthetases, we carried out conformational and mutational analyses of the conserved residues, addressing: i) the importance of the BM-motif glycine, and ii) the role of other highly conserved residues around the BM-motif glycine.


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Fig. 1.   A, BM-motif: comparison of bifunctional PAPS synthetase sequences from mouse, human, and Cavia porcellus with several monofunctional APS kinase sequences from lower organisms. Conserved residues are shown in the consensus sequence; h is a conserved hydrophobic position, and x is a nonconserved position. B, comparison of the BM-motif sequence from mouse bifunctional PAPS synthetase with the BM-motif-like region found in the regulatory domain of monofunctional ATP sulfurylases from P. chrysogenum and S. cerevisiae.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of Mutant Constructs-- Our previous extensive mutational analyses (9, 11) were performed on the SK1 isoform protein, thus SK1 was used for functional analysis of the BM-motif (residues Leu86-Lys97). Site-directed mutations in SK1 cDNA were generated by insertion of double-stranded oligonucleotides. Briefly, in a ProEx plasmid obtained from a ProEx/SK1 construct, two unique restriction enzyme sites, Bsm1 and Bsu36I, flanking the BM-motif were used to remove the normal cDNA sequence and to ligate in the mutated BM-motif. The mutational inserts altering the BM-motif also contained an engineered translationally silent restriction site, AvrII, which is not present in the original ProEx/SK1 cDNA construct. Making use of the AvrII site, the colonies containing the mutated BM-motif constructs were identified by double digestion of plasmid DNA with AvrII and XhoI; positive clones were verified by DNA sequencing.

Expression and Purification of Protein-- BL21 DE3 (codon plus) from cells transformed with normal or mutated plasmid constructs were grown in LB/ampicillin/chloamphenicol liquid culture at 37 °C to an A600 of 1.0. Following induction with 0.1 M isopropyl-thio-beta -D-galactoside for 4 h, the cultures were centrifuged at 9000 × g for 8 min, and the pellet was resuspended in 4 volumes of 50 mM Tris-HCl, 5 mM beta -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (freshly prepared), pH 8.5 at 4 °C. The suspension was sonicated, the cellular debris removed by ultracentrifugation at 15,000 × g, the supernatant loaded onto a nickel-nitrilotriacetic acid-agarose resin column. The column was then washed with 7 column volumes of Buffer A (20 mM Tris-HCl, 100 mM KCl, 5 mM beta -mercaptoethanol, 20 mM imidazole, 10% glycerol, pH 8.5 at 4 °C), 7 column volumes of Buffer B (20 mM Tris-HCl, 1 M KCl, 5 mM beta -mercaptoethanol, 10% glycerol, pH 8.5 at 4 °C), and again with 7 column volumes of Buffer A. Protein was eluted with buffer C (20 mM Tris-HCl, 100 mM KCl, 200 mM imidazole, 5 mM beta -mercaptoethanol, 10% glycerol, pH 8.5 at 4 °C). The eluted protein was dialyzed against 20 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.8, buffer. Protein concentration was determined using the Bradford assay.

Fluorescence Substrate Binding Experiments-- The concentration of protein used in fluorescence experiments was ~200 µg/ml. To eliminate condensation of moisture on the fluorescence sample cell, the experiments were performed at 10 °C. Tryptophan fluorescence emission was recorded in the 305-400 nm range with an excitation wavelength of 295 nm. Substrate binding experiments were performed at the peak emission wavelength for each substrate (342 nm for LAPSK2 and 338 nm for BM-LAPSK2). Buffer fluorescence was subtracted from sample fluorescence before determining binding constants.

Enzyme Assays-- The purified recombinant protein was assayed for reverse ATP sulfurylase, APS kinase, and overall PAPS synthetase activities using protocols as described earlier (9). All assays were performed at least three times; data are presented as the average of individual results. For low activity mutants, a higher stock concentration of protein (400 µg/ml) was tested to differentiate the specific activities.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Structural and Substrate Binding Analysis-- To investigate the impact of the bm mutation on structural and substrate-binding properties of the kinase domain, fluorescence intensity experiments, which are highly sensitive to the molecular environment of tryptophan residues and allow conformational changes due to substrate binding to be detected directly, were performed. However, since there are 11 and 10 tryptophans in SK1 and SK2, respectively, and two/three substrate binding pockets, it is possible that an increase in fluorescence in one portion of the protein may mask a decrease in fluorescence from another portion resulting in no net change in signal, or that the obtained fluorescence signal may be difficult to de-convolute for the bifunctional protein. Thus, the expressed kinase domains of SK1 (LAPSK1, with two tryptophans) and SK2 (LAPSK2, with only one tryptophan), as well as BM-LAPSK2, the kinase domain of SK2 with bm mutation (G79R), were used for these studies.

Buffer-subtracted fluorescence of the three proteins is shown in Fig. 2. Expectedly, LAPSK1, which contains two tryptophans, exhibited higher fluorescence intensity compared with LAPSK2, which contains only one. However, it was surprising that BM-LAPSK2 exhibited 60% loss of fluorescence intensity compared with LAPSK2, thus indicating that i) Trp47 in LAPSK2 is conformationally coupled to Gly79 and ii) there is significant structural change in the kinase domain due to the bm mutation. To assess the effect of the bm mutation on substrate binding, the binding constants for ATP and APS were determined for LAPSK2 and BM-LAPSK2. From the binding curves shown for LAPSK2 (Fig. 3, A and B) KD(ATP) was determined to be 0.3 µM and KD(APS) was 0.13 µM., while for BM-LAPSK2 (Fig. 3, C and D) KD(ATP) was 28.5 µM and KD(APS) was 5.4 µM, suggesting that BM-LAPSK2 binds both ATP and APS poorly. Thus, a decrease in substrate binding caused by the bm mutation may account for the loss of kinase and overall activities in BM-SK2.


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Fig. 2.   Buffer-subtracted intrinsic tryptophan fluorescence spectra of kinase domains of SK1 (LAPSK1), SK2 (LAPSK2), and SK2 with bm mutation (BM-LAPSK2). Excitation wavelength was set to 295 nm, and fluorescence emission was measured between 305-400 nm.


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Fig. 3.   Substrate binding experiments for LAPSK2 and BM-LAPSK2. A, ATP titration with LAPSK2. B, APS titration with LAPSK2. C, ATP titration with BM-LAPSK2. D, APS titration with BM-LAPSK2. Nanomolar (×10) concentration of ATP and APS was used in this experiment.

DGD-turn Mutants-- To explore the structural and functional contribution of the BM-motif (residues 86-97) to SK1 activity, we undertook site-specific mutagenesis studies targeting Gly88, the flanking aspartates Asp87 and Asp89 of the DGD-turn, and the highly conserved residues flanking the DGD-turn in addition to two residue-order switch mutants. Mutations of the DGD-turn residues included D87A, D87R, D87E, G88A, G88R, G88D, D89A, D89R, and D89E, designed to investigate structural aspects (i.e. glycine as a flexible amino acid) as well as charge requirements (i.e. negative charge due to aspartic acid residues). Activity data for wild-type SK1 and the mutant enzymes are presented in Tables I and II.

                              
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Table I
DGD-loop mutants

                              
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Table II
Switch mutants and other conserved residue mutants

Although G88A exhibited reverse sulfurylase activity comparable to wild-type, overall (75%) and kinase (69%) residual activities were reduced, suggesting that a flexible amino acid at residue 88 is required for optimal kinase activity. G88R, a mutation analogous to that in the brachymorphic mouse, was designed to test whether SK1 behaves similarly to SK2. As observed for the naturally occurring bm mutation in SK2, the G88R mutation in SK1 leads to complete loss of overall (0.5%) and kinase (4.6%) activities. The relative overall activity of G88D, designed to test the negative charge requirement for the turn region, was reduced to 0.2% and kinase activity to 4.4%. In reverse sulfurylase assays, both G88R and G88D showed activities comparable to wild type.

N-terminal to the bm glycine, an aspartate analogous to residue Asp87 is strictly conserved among all the monofunctional APS kinases and bifunctional PAPS synthetases (Fig. 1A). Based on the APS kinase crystal structure from Penicillium chrysogenum, MacRae et al. (12) predicted that a negative charge presented by Asp87 may help bind Mg2+, which in turn presents a positive charge to ATP/APS. Removal of the negative charge by mutating Asp87 to alanine resulted in decreased overall (0.6%) and kinase (6.7%) activities. Likewise, D87R, with the charge reversed, had an activity profile comparable D87A. D87E, which retains the negative charge but extends the side chain by one C-C bond (1.5 Å), was designed to test the tightness and the charge requirements of the substrate binding pocket. Unexpectedly, the relative overall activity for the charge-conserved mutation, D87E, was observed to be only 2.2%, while, there was a slight improvement in kinase activity (18%) compared with D87A or D87R. None of the Asp87 mutants exhibited any loss of reverse sulfurylase activity.

Asp89, the residue C-terminal to the BM-motif glycine, is located at the N terminus of the alpha -helix that follows the DGD-turn, based on the crystal structure of APS kinase from P. chrysogenum (12). Although the D89A and D89R mutant proteins both exhibited reverse sulfurylase activity comparable to that of wild-type SK1, they show <0.5% residual overall and <5% kinase activities, suggesting that the negative charge contributed by Asp89 is also essential for a functional APS kinase. Designed to assess the structural flexibility and residue stringency for this position, the D89E mutant showed an improvement in overall (9%) and kinase (23%) activities relative to D89A and D89R, but still indicates a size restriction in this position.

Residue-order Switch Mutants-- To determine whether there is functional restriction on the location of the aspartates, a set of residue-order switch mutations, LD86DL and DN89ND, were designed in which the critical aspartates were moved one residue further away from the DGD-turn on either the N-terminal or C-terminal side of the central glycine. The residual overall (4.9%) and kinase (5.3%) activities of the LD86DL protein suggest that not only is the presence of aspartate necessary for activity, but that the active site is rigid and cannot easily adapt to an altered chemical and electrostatic environment by a shift in the position of the acidic residues. Surprisingly, LD86DL, although mutated in the kinase domain, also had a 60% loss of reverse sulfurylase activity, suggesting involvement of these residues in both the sulfurylase and kinase activities. When residues Asp89 and Asn90 were interchanged in DN89ND, complete loss of kinase (1.5%) and overall (0.9%) activities was observed, again indicating that the BM-motif is very rigid and that this alteration is detrimental to both exogenous and endogenous APS utilization. In contrast to LD86DL, the DN89ND protein maintained reverse sulfurylase activity comparable to wild type.

Conserved Flanking Residue Mutations-- The third set of mutants included N90A, N90Q, R92A, and K97A, which are highly conserved and are located near the C terminus of the DGD-turn (Fig. 1). Despite the strong conservation of Asn90 and its proximity to the DGD-loop, deletion of the side chain amide functionality in N90A resulted in 67% residual overall and kinase activities, N90Q tested whether substituting another amide-presenting side chain residue could prevent losses in overall or kinase activity. Interestingly, the residual overall (64%) and kinase (69%) activities were similar to those for N90A with retention of reverse sulfurylase activity comparable to that of wild-type SK1. In contrast, R92A exhibited complete loss of overall (1.1%) and kinase (0.9%) activities, while reverse sulfurylase activity was normal. Lastly, Lys97, which is also highly conserved and present in the loop following the alpha -helix (residue 88-94), exhibited all three activities comparable to those of wild-type SK1, suggesting that Lys97 is not essential in SK function.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The BM-motif, which is comprised of a DGD-turn and flanking conserved residues, was initially identified through the mutation discovered in SK2 cDNA cloned from the brachymorphic mouse (7). Sequence comparisons show that the BM-motif is highly conserved among several bifunctional PAPS synthetases and monofunctional APS kinases. Analysis of the BM-motif by tryptophan fluorescence experiments provides the first evidence that the region in SK2 containing the bm mutation (G79R) is important for the structure of the protein and that alteration in conformation associated with the bm mutation accounts for the loss of kinase activity. Furthermore, substrate binding fluorescence experiments show that LAPSK2 exhibits tight binding to APS and ATP, while the bm mutation in LAPSK2 leads to significant reductions in ATP (100-fold) and APS (50-fold) binding, indicating that Gly79 is located in the vicinity of both the ATP and APS binding sites and that the peptide region around Gly79 is a critical component of the PAPS synthetase active site(s).

A systematic mutagenesis analysis of the BM-motif showed that, as observed in mutant SK2 from the brachymorphic mouse, changing the SK1 DGD-turn glycine to arginine (G88R) produces a nearly inactive kinase and subsequent loss of overall activity (7). Substitution of a positive charge at this position could result in displacement of the MgATP-magnesium or misalignment of the gamma -phosphate of ATP, leading to either weaker or complete loss of ATP binding and thereby causing the observed decreases in kinase and overall activities. Even recombinant enzyme with a G88A substitution, which replaces a neutral, flexible amino acid with a neutral, good alpha -helix-forming residue, exhibited some reduction (30%) in overall and kinase activities, possibly due to an increase in rigidity of the active site via N-terminal extension of the alpha -helix that follows the DGD-turn, thus causing misalignment of critical aspartates around Gly88. Similar structural consequences may induce the activity loss observed for the G88R and G88D mutant enzymes, as both arginine and aspartic acid residues have higher alpha -helix propensities than glycine. Furthermore, while the presence of negatively charged residues is a requirement for DGD-turn function, the number and spacing of acidics is critically important since the G88D mutant exhibited a severe loss (0.2%) of overall activity.

The results of multiple mutations at Asp87 and Asp89 strongly suggest an absolute requirement for both acidics. Furthermore, the low overall activity observed for glutamic acid substitutions at Asp87 and Asp89 indicate that the active site is not very flexible and that the correctly sized residue side chains are important for internal transfer of APS. These possibilities are partly supported by the findings of MacRae et al. (12), which predicted a role for Asp87 in ATP binding, while no such role was predicted for Asp89. However, structural comparison of P. chrysogenum APS kinase with substrate-bound structures of guanylate kinase (13) and 6-phosophofructo-2-kinase (14) suggest that APS is placed between the P-loop, FISP-motif, and the alpha -helix C-terminal of the DGD-loop. Furthermore, during the course of our functional analysis of the BM-motif, MacRae et al. crystallized P. chrysogenum ATP sulfurylase in the presence of APS (15), and showed that APS was bound to the sulfurylase active site as well as to the C-terminal kinase-like regulatory domain. As we predicted, this crystal structure shows residue Asp434, analogous to Asp89 in SK1, interacting with the 3'-OH of APS, thereby aiding in proper alignment of APS for phosphoryl transfer. Thus, Asp89 may be participating in internal transfer and reactive alignment of endogenously generated APS in the kinase active site, whereas Asp87 may be involved in the ATP binding and phosphoryl transfer step of the kinase reaction.

As mentioned, monofunctional ATP sulfurylases of P. chrysogenum and Saccharomyces cerevisiae contain inactive C-terminal APS-kinase-like regulatory domains (Fig. 1B); however, the kinase DGD-loop is replaced by LGD-loop in these sulfurylase regulatory domains. In addition to the presence of a leucine at residue 432 (analogous to Asp87 in SK1), the P-loop in the kinase-like domain is changed from GLSASGKS to GYMNSGKD. Independent studies of bifunctional SK1 (9) and monofunctional APS kinase (16) have shown that a single mutation in the key residues of the P-loop is sufficient to inactivate the kinase. Loss of kinase activity in our D87A mutant supports the hypothesis put forward by MacRae et al. (15, 16) that lack of APS kinase activity in the ATP sulfurylase regulatory domain is due both to the altered P-loop and the absence of an aspartate at residue 432.

Additional conserved residues, C-terminal to the DGD-turn, also contribute to PAPS synthetase activity. Based on the P. chrysogenum APS kinase structure, Asn90 is expected to be present on the face opposite the substrate binding pocket, Arg92 is on the substrate binding face of the alpha -helix, and Lys97 on the loop following the alpha -helix. Equal and low (30%) residual activity for N90A and N90Q suggests that Asn90 may be playing a structural role via interactions with other residues present on the non-substrate binding face of the alpha -helix to allow the correct orientation of critical residues on the substrate binding face. The presence of Arg92 on the substrate binding face and a near-complete loss of the overall and kinase activities for R92A suggest that it could be participating in the binding of APS or in the alignment of Asp89 via a salt bridge. In fact, the crystal structure of P. chrysogenum ATP sulfurylase, which shows that one of the specific interactions between APS and the allosteric domain is a bidentate salt linkage between the guanidino head of Arg437 and the phosphosulfate moiety of APS, confirms our prediction that Arg92 is binding to APS.

In our previous study of the P-loop (9) we showed that mutation of the kinase domain residue Gly59 to alanine disrupted sulfurylase activity. Also, in our subsequent analysis of the PP-loop (11) which is located in the sulfurylase domain, we identified two residues, His506 and Asp523, that when mutated to alanine resulted in altered kinase activity. Similar cross-reactivity behavior was observed in the present study for LD86DL, a switch mutant designed to probe the strictness of the spatial constraint on the critical aspartate, Asp87, and the flexibility of the active site. Lastly, it is interesting to observe that in all of the DGD-turn mutants the loss in overall activity is greater than the loss in kinase activity, suggesting that the conversion of endogenous APS, an intermediate in the overall reaction to PAPS, is affected by the BM-motif mutations to a greater degree than is the conversion of exogenous APS, a substrate in the kinase reaction. Thus, in addition to participating in the kinase reaction, the BM-motif may be interacting with the sulfurylase domain and playing a role in the transfer of APS from the sulfurylase to the kinase active site. The hypothesis of less efficient utilization of endogenous APS in the bm mutant enzyme is consistent with results from our previous study where we demonstrated that SK2 isolated from brachymorphic mice exhibits a channeling defect (4). Further studies to understand the channeling of APS by the recombinant bm-like SK1 are in progress.

In summary, our past work sought to identify the components of the APS kinase active site by analyzing the contribution of three functional motifs: the P-loop (9), the FISP-motif (10) and the PAPS binding motif (10). With the elucidation of the mutation in the bm mouse (7), we have now identified a fourth component of the kinase active site, the BM-motif. Our mutational analysis of the BM-motif shows the functional importance of several highly conserved residues of this region in enzyme catalysis, endogenous APS transfer, and in formation of a rigid active site and as well provides a rationale for the lack of kinase activity in the kinase-like regulatory domain of P. chrysogenum ATP sulfurylase.

    ACKNOWLEDGEMENTS

We thank Dr. Youli Wang for help in constructing mutant enzymes, James Mensch for helpful comments during the course of this study, and Daniel Vatner for help in sequence comparison.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HD-17332 and a fellowship from the Markey Foundation (to B. S.).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.

Dagger To whom correspondence should be addressed: Dept. of Pediatrics, University of Chicago, 5841 S. Maryland Ave., MC 5058, Chicago, IL 60637. Tel.: 773-702-6426; Fax: 773-702-9234; E-mail: n-schwartz@uchicago.edu.

Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M206688200

    ABBREVIATIONS

The abbreviations used are: PAPS, 3'-phosphate 5'-phosphosulfate; APS, adenosine 5'-phosphosulfate; bm, brachymorphic; PP, pyrophosphate; SK1, mouse PAPS synthetase isoform 1; SK2, mouse PAPS synthetase isoform 2.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Schwartz, N. B., Lyle, S., Ozeran, J. D., Li, H., Deyrup, A., Ng, K., and Westley, J. (1998) Chem. Biol. Interact. 109, 143-151[CrossRef][Medline] [Order article via Infotrieve]
2. Sugahara, K., and Schwartz, N. B. (1982) in Proceedings of the 6th International Symposium on Glycoconjugate (Yamakawa, T. , Oswa, T. , and Handa, S., eds) , pp. 493-495, Science Society Press, Tokyo, Japan
3. Lyle, S., Ozeran, J. D., Stanzak, J., Westley, J., and Schwartz, N. B. (1994) Biochemistry 33, 6822-6827[Medline] [Order article via Infotrieve]
4. Lyle, S., Stanzak, J., Westley, J., and Schwartz, N. B. (1995) Biochemistry 34, 940-945[Medline] [Order article via Infotrieve]
5. Rusiniak, M. E., O'Brien, E. P., Novak, E. K., Barone, S. M., McGarry, M. P., Reddington, M., and Swank, R. T. (1996) Mamm. Genome 7, 98-102[CrossRef][Medline] [Order article via Infotrieve]
6. Schwartz, N. B. (1983) Limb Development and Regeneration , Vol. B , pp. 97-103, A. R. Liss, New York
7. Kurima, K., Warman, M. L., Krishnan, S., Domowicz, M., Krueger, R. C., Jr., Deyrup, A., and Schwartz, N. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8681-8685[Abstract/Free Full Text]
8. Li, H., Deyrup, A., Mensch, J., Domowicz, M., Konstantinidis, A., and Schwartz, N. B. (1995) J. Biol. Chem. 270, 29453-29459[Abstract/Free Full Text]
9. Deyrup, A. T., Krishnan, S., Cockburn, B. N., and Schwartz, N. B. (1998) J. Biol. Chem. 273, 9450-9456[Abstract/Free Full Text]
10. Deyrup, A. T. (1997) Pathology , University of Chicago, Chicago, IL
11. Deyrup, A. T., Singh, B., Krishnan, S., Lyle, S., and Schwartz, N. B. (1999) J. Biol. Chem. 274, 28929-28936[Abstract/Free Full Text]
12. MacRae, I. J., Segel, I. H., and Fisher, A. J. (2000) Biochemistry 39, 1613-1621[CrossRef][Medline] [Order article via Infotrieve]
13. Stehle, T., and Schulz, G. E. (1992) J. Mol. Biol. 224, 1127-1141[Medline] [Order article via Infotrieve]
14. Hasemann, C. A., Istvan, E. S., Uyeda, K., and Deisenhofer, J. (1996) Structure 4, 1017-1029[Medline] [Order article via Infotrieve]
15. MacRae, I. J., Segel, I. H., and Fisher, A. J. (2001) Biochemistry 40, 6795-6804[CrossRef][Medline] [Order article via Infotrieve]
16. MacRae, I. J., Rose, A. B., and Segel, I. H. (1998) J. Biol. Chem. 273, 28583-28589[Abstract/Free Full Text]


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