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

Bhawani Singh, Nancy B. Schwartz
2002 Journal of Biological Chemistry  
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
more » ... evealed 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 Asp 87 and Asp 89 , 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 Asn 90 , Arg 92 , and Lys 97 . 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. 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 bifunc-tional 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 4 2Ϫ and APS kinase, which phosphorylates APS in the presence of another molecule of ATP to form PAPS. 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 (Gly 79 ) is very high; however, no function had been established for this region. Based on the sequence alignment shown in
doi:10.1074/jbc.m206688200 pmid:12414806 fatcat:zupzohhlrbavtkzzqwkoidwh5u