Molecular and catalytic properties of Arabidopsis thaliana adenylyl sulfate (APS)-kinase.

A cDNA clone (Atakn1) from Arabidopsis thaliana encoding APS-kinase (EC 2.7.1.25) was investigated for structural and catalytic properties of the gene product. Recombinant his10-AtAkn1 formed PAPS at a Vmax of 7.35 U x mg(-1). The Km for APS was 0.14 microM and for ATP 147 microM. APS caused a severe substrate inhibition (K(i) 4.5 microM). The type of inhibition is uncompetitive with respect to MgATP. High ionic strength and reducing thiols stabilized the enzyme activity. Plant APS-kinase is regulated in vitro by the redox charge with thioredoxin as essential activator. Mutagenesis of a serine in S182C and S182F presumed to be involved in the transfer of the phosphoryl group had no effect upon catalytic activity. Using a yeast two-hybrid system with AtAkn1 as bait, an interacting clone was detected from a cDNA library of A. thaliana cv. Columbia that codes for an APS-kinase iso-form (Atakn2). Complementation of APS-kinase-deficient Saccharomyces cerevisiae met14 showed that AtAkn2 is functionally active as APS-kinase. It was immunologically related to AtAkn1 and presumably represents a plastidal iso-form of the plant APS-kinase gene family.

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