Cloning and characterization of a second acid phosphatase from Sinorhizobium meliloti strain 104A14

Sinorhizobium meliloti has two nonspecific periplasmic acid phosphatases. T he NapD enzyme has been previously described, and a second acid phosphatase , NapE, is described in this report. NapE was partially purified from an S. meliloti napD mutant and characterized with respect to molecular mass and substrate range. As predicted from SDS-PAGE analysis, the subunit molecular mass of NapE is approximately 35.8 kDa and gel filtration experiments esti mated the native molecular mass to be approximately 70 kDa, indicating that the active enzyme is a homodimer. NapE demonstrated significant activity w ith p-nitrophenyl phosphate, phenyl phosphate, and alpha -naphthyl-phosphat e. The pH optimum was between 4.5 and 5.0. The gene encoding NapE was also sequenced and the inferred amino acid sequence from the predicted ORF was f ound to be 60% identical and 75% similar to that encoded by napD. An S. mel iloti napE mutant was constructed and assessed for symbiotic competence. Th is mutant did not differ from the wild-type parent strain in nodulation and symbiotic efficiency.