Identification and functional characterization of the novel BM-motif in the murine phosphoadenosine phosphosulfate (PAPS) synthetase.
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 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.
Singh
Bhawani
B
Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA.
Schwartz
Nancy B
NB
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013430:Sulfate Adenylyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
D007527:Isoenzymes; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D014364:Tryptophan; C022322:PAPS synthetase; D013430:Sulfate Adenylyltransferase