Arabidopsis thaliana Adenosine 5'-Phosphosulfate Kinase: Structure, Function and Regulation
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thesis / dissertation description
In plants, the essential nutrient sulfur is partitioned between primary (reductive) and secondary (sulfotransfer) pathways, generating a wide array of sulfonated metabolites. APS reductase (APSR) kinetically controls entry into the reductive assimilatory pathway, and has been characterized extensively. APS kinase (APSK) kinetically controls entry into the secondary assimilatory pathway that supports the synthesis of multiple sulfonated metabolites. Neither the biochemical regulation of APSK nor the control of sulfur partitioning between the two branches of the sulfur assimilatory pathway in plants are understood. This work identifies and examines the molecular mechanisms by which APSK activity is regulated in vitro.To understand how APSK functions, x-ray crystallography was used to determine the three-dimensional structure of the enzyme from Arabidopsis thaliana. The 1.8 Å resolution crystal structure of AtAPSK in complex with AMP-PNP, Mg2+, and APS provided the first view of the Michaelis complex for this enzyme and revealed the presence of an intersubunit disulfide bond between Cys86 and Cys119. Functional analysis of AtAPSK demonstrated that reduction of Cys86-Cys119 resulted in a 17-fold higher kcat/Km and a 15-fold increase in Ki for substrate inhibition by APS compared to the oxidized enzyme. The C86A/C119A mutant was kinetically similar to the reduced wild-type enzyme. Gel- and activity-based titrations indicated that the midpoint potential of the disulfide in AtAPSK was comparable to that observed in APS reductase. Both cysteines are invariant among the APSK from plants, but not other organisms, which suggests redox-control as a novel regulatory feature of the plant APSK. Based on structural, functional, and sequence analyses, we proposed that the redox-sensitive APSK evolved after bifurcation of the sulfur assimilatory pathway in the green plant lineage and that changes in redox environment resulting from oxidative stresses may affect partitioning of APS into the primary and secondary thiol metabolic routes by having opposing effects on APSK and APS reductase in plants.For catalysis, APSK coordinates the addition of structurally similar phosphonucleotides, but competing models for the enzyme have been proposed. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e., ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp136, which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.We also examined the role of the AtAPSK N-terminal domain, which is the site of redox regulation through the Cys86-Cys119 linkage, on nucleotide binding and substrate inhibition. Titrations of reduced and oxidized AtAPSK with nucleotides clearly show that in the absence of either ATP or ADP, APS binds with significantly higher affinity to the latter, while the affinity for ATP and ADP decreased. A model is proposed in which disulfide bond formation promotes oxidized AtAPSK apoenzyme to adopt a conformation analogous to that of reduced AtAPSK in complex with ADP, which mimics an inhibitory effect. These results also suggest that substrate inhibition results from formation of both APSK*APS and APSK*ADP*APS complexes and provides a molecular basis for how redox regulation of AtAPSK affects activity.