Abstract
Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine 5'-phosphosulfate (APS) from sulfate and ATP. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substrate-binding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3'-phosphoadenosine 5'-phosphosulfate synthetase. Steady-state kinetic analysis of 20 G. max ATP sulfurylase point mutants suggests a reaction mechanism in which nucleophilic attack by sulfate on the α-phosphate of ATP involves transition state stabilization by Arg-248, Asn-249, His-255, and Arg-349. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway.
Highlights
ATP sulfurylase catalyzes the energetically unfavorable formation of adenosine 5Ј-phosphosulfate in plant sulfur assimilation
ATP sulfurylase activity and sulfate levels were determined in leaves of accessions representative of each haplotype grown for 5 weeks in a controlled environment room under short days
ATP sulfurylase activity and sulfate were measured in A. thaliana Col-0 and atps1 mutant grown at the same conditions
Summary
ATP sulfurylase catalyzes the energetically unfavorable formation of adenosine 5Ј-phosphosulfate in plant sulfur assimilation. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substratebinding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3-phosphoadenosine 5-phosphosulfate synthetase. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway
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