Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 ha s been cloned, sequenced, and expressed in an active form. A 600-bp PCR pro duct generated using primers designed from amino acid sequence data was use d to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfati ng sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase pro protein was identified, and the deduced 517-amino-acid protein minus its si gnal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors . However, cloning the gene into a Bacteroides expression vector did produc e active sulfatase. This is the first mucin-desulfating sulfatase to be seq uenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direc tion. Its sequence has close homology to iron-sulfur proteins that posttran slationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary fo r enzymatic activity.