The molecular class C acid phosphatase of Chryseobacterium meningosepticum (OlpA) is a broad-spectrum nucleotidase with preferential activity on 5 '-nucleotides

摘要

The olpA gene of Chryseobacterium meningosepticum, encoding a molecular class C phosphatase, was cloned and expressed inEscherichia coli. The gene encodes a 29-kDa polypeptide containing an amino-terminal signal peptide typical of bacterial membranelipoproteins. Expression in E. coli results in a functional product that mostly partitions in the outer membrane. A secreted soluble OlpAderivative (sOlpA) lacking the N-terminal cysteine residue for lipid anchoring was produced in E. coli and purified by means of two steps ofion exchange chromatography. Analysis of the kinetic parameters of sOlpA with several organic phosphoesters revealed that the enzyme wasable to efficiently hydrolyze nucleotide monophosphates, with a strong preference for 5V-nucleotides and for 3V-AMP. The enzyme was alsoable to hydrolyze sugar phosphates and h-glycerol phosphate, although with a lower efficiency, whereas it was apparently inactive againstnucleotide di- and triphosphates, diesters, and phytate. OlpA, therefore, can be considered a broad-spectrum nucleotidase with preference for5V-nucleotides. Its functional behaviour exhibits differences from that of the Haemophilus influenzae OMP P4 lipoprotein, revealingfunctional heterogeneity among phosphatases of molecular class C.