Genetic and biochemical characterization of a protein phosphatase with dual substrate specificity in Streptomyces coelicolor A3(2)

A gene encoding a protein phosphatase (SppA) with a phosphoesterase motif, which was predicted by the genome project of the Gram-positive bacterium St reptomyces coelicolor A3(2), was cloned by PCR in pET32a(+) and expressed i n Escherichia coli. SppA fused to thioredoxin (TRX-SppA) showed distinct he at-stable phosphatase activity toward p-nitrophenyl phosphate with optimal pH 8.0 and optimal temperature 55 degreesC. Mn2+ greatly enhanced enzyme ac tivity, as is found with other protein Ser/Thr phosphatases. TRX-SppA was n ot inhibited by sodium orthovanadate or okadaic acid, both of which are kno wn to be specific inhibitors of protein phosphatases. TRX-SppA showed phosp hatase activity toward not only phosphoThr (pThr) and pTyr but also oligope ptides containing pSer, pThr, and pTyr, indicating that SppA is a protein p hosphatase with dual substrate specificity. Disruption of the chromosomal s ppA gene resulted in severe impairment of vegetative growth. All of these o bservations show that SppA, a protein phosphatase with dual specificity, pl ays an important, but not essential, role in vegetative growth of S. coelic olor A3(2). The presence of a single copy of sppA in all the 13 Streptomyce s species examined, as determined by Southern hybridization, suggests a com mon role of SppA in general in Streptomyces species. (C) 2000 Elsevier Scie nce B.V. All rights reserved.