THE ISOLATION AND CHARACTERIZATION OF CDNA-ENCODING THE MOUSE BIFUNCTIONAL ATP SULFURYLASE-ADENOSINE 5'-PHOSPHOSULFATE KINASE

Biosynthesis of the activated sulfate donor, adenosine 3'-phosphate 5' -phosphosulfate, involves the sequential action of two enzyme activiti es: ATP sulfurylase, which catalyzes the formation of adenosine 5'-pho sphosulfate (APS) from ATP and free sulfate, and APS kinase, which sub sequently phosphorylates APS to produce adenosine 3'-phosphate 5'-phos phosulfate. Oligonucleotide primers were derived from a human infant b rain-expressed sequence tag putatively encoding a portion of APS kinas e. Using these primers, reverse transcriptase-polymerase chain reactio n was performed on mRNA from neonatal normal mice resulting in amplifi cation of a 127-bp DNA fragment. This fragment was subsequently used t o screen a mouse brain lambda gt11 cDNA library, yielding a 2.2-kb clo ne. Primers were designed from the 5'-end of the 2.2-kb clone, and 5'- rapid amplification of cDNA ends was used to obtain the translation st art site. Sequence from the overlapping clones was assembled into a 24 75-bp composite sequence, which contains a single open reading frame t hat translates into a 624-deduced amino acid sequence. Northern blots of total RNA from neonatal mice yielded a single message species at ap proximately 3.3 kb. Southern blot of genomic DNA digested with several restriction enzymes suggested the gene is present as a single copy. C omparison against sequence data bases suggested the composite sequence was a fused sulfurylase-kinase product, since the deduced amino acid sequence showed extensive homology to known separate sequences of both ATP sulfurylase and APS kinase from several sources. The first 199 am ino acids corresponded to APS kinase sequence, followed by 37 distinct amino acids, which did not match any known sequence, followed by 388 amino acids that are highly homologous to known ATP sulfurylase sequen ces. Finally, recombinant enzyme expressed in COS-1 cells exhibited bo th ATP sulfurylase and APS kinase activity.