A CDNA FOR ADENYLYL SULFATE (APS)-KINASE FROM ARABIDOPSIS-THALIANA

A cDNA done with an open reading frame of 831 nucleotides was isolated from a lambda ZapII-library of Arabidopsis thaliana. The nucleotide s equence of the cDNA is homologous to the APS-kinase genes from enterob acteria, diazotrophic bacteria, and yeast: Escherichia coli (cys C: 53 .2%), Rhizobium meliloti (nod Q: 52.6%), and Saccharomyces cerevisiae (met 14: 57.1%). The polypeptide deduced from the plant APS-kinase cDN A is comprised of 276 amino acid residues with a molecular weight of 2 9 790. It contains an N-terminal extension of 77 amino acids. This ext ension includes a putative transit peptide of 37 residues separated fr om the core protein by a VRACV processing site for stromal peptidase; a molecular weight of 26 050 is predicted for the processed protein. T he relatedness between bacterial, fungal and plant APS-kinase polypept ides ranges from 47.5% (E. coli), 55.4% (S. cerevisiae), 52.6% (R. mel iloti), and 50.3% (Azospirillum brasilense). The plant polypeptide con tains eight cysteine residues; two cysteines flank a conserved purine nucleotide binding domain: GxxxGK, Also conserved are a serine-182 as a possible phosphate transferring group and a K/LARAGxxxxFTG motif des cribed for PAPS dependent enzymes. The identity of the gene was confir med by analyzing the function of the gene product. The putative transi t peptide was deleted by PCR and the truncated gene was expressed in a pTac1 vector system. A polypetide of MW 25761 could be induced by IPT G. The gene product was enzymatically active as APS-kinase. It produce d PAPS from APS and ATP - the absence of ATP but supplemented with thi ols, the APS-kinase reacted as APS-sulphotransferase. APS-sulphotransf erase is not a separate enzyme but identical with APS-kinase.