The isolation and characterization of cDNA encoding the mouse bifunctional ATP sulfurylase-adenosine 5′-phosphosulfate kinase

Abstract

Biosynthesis of the activated sulfate donor, adenosine 3′-phosphate 5′-phosphosulfate, involves the sequential action of two enzyme activities: ATP sulfurylase, which catalyzes the formation of adenosine 5′-phosphosulfate (APS) from ATP and free sulfate, and APS kinase, which subsequently phosphorylates APS to produce adenosine 3′-phosphate 5′-phosphosulfate. Oligonucleotide primers were derived from a human infant brain-expressed sequence tag putatively encoding a portion of APS kinase. Using these primers, reverse transcriptase-polymerase chain reaction was performed on mRNA from neonatal normal mice resulting in amplification of a 127-bp DNA fragment. This fragment was subsequently used to screen a mouse brain λgt11 cDNA library, yielding a 2.2-kb clone. 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 start site. Sequence from the overlapping clones was assembled into a 2475-bp composite sequence, which contains a single open reading frame that translates into a 624-deduced ami no acid sequence. Northern blots of total RNA from neonatal mice yielded a single message species at approximately 3.3 kb. Southern blot of genomic DNA digested with several restriction enzymes suggested the gene is present as a single copy. Comparison 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 amino 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 sequences. Finally, recombinant enzyme expressed in COS-1 cells exhibited both ATP sulfurylase and APS kinase activity.