Isolation, Cloning, and Expression of an Acid Phosphatase Containing Phosphotyrosyl Phosphatase Activity from Prevotella intermedia

Abstract

A novel acid phosphatase containing phosphotyrosyl phosphatase (PTPase) activity, designated PiACP, from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease, has been purified and characterized. PiACP, a monomer with an apparent molecular mass of 30 kDa, did not require divalent metal cations for activity and was sensitive to orthovanadate but highly resistant to okadaic acid. The enzyme exhibited substantial activity against tyrosine phosphate-containing peptides derived from the epidermal growth factor receptor. On the basis of N-terminal and internal amino acid sequences of purified PiACP, the gene coding for PiACP was isolated and sequenced. The PiACP gene consisted of 792 bp and coded for a basic protein with an M r of 29,164. The deduced amino acid sequence exhibited striking similarity (25 to 64%) to those of members of class A bacterial acid phosphatases, including PhoC of Morganella morganii, and involved a conserved phosphatase sequence motif that is shared among several lipid phosphatases and the mammalian glucose-6-phosphatases. The highly conservative motif HCXAGXXR in the active domain of PTPase was not found in PiACP. Mutagenesis of recombinant PiACP showed that His-170 and His-209 were essential for activity. Thus, the class A bacterial acid phosphatases including PiACP may function as atypical PTPases, the biological functions of which remain to be determined.

Exploiting Acid Phosphatases in the Synthesis of Phosphorylated Monoalcohols and Diols

“…Acid phosphatase AphA‐St from Salmonella typhimurium LT221 was selected due to its phosphotransferase activity on alcohols using p ‐NPP as donor 22. PiACP from Prevotella intermedia 23 was selected based on high sequence similarity with robust PhoC‐Mm developed for nucleoside phosphorylation 24. All enzymes except phytase were overexpressed in E. coli and subsequently purified to homogeneity by using His‐tag or Strep‐tag techniques (Figure S1).…”

A novel exopolysaccharide from a clinical isolate of Prevotella nigrescens: purification, chemical characterization and possible role in modifying human leukocyte phagocytosis

“…The pathogenic potential of P. nigrescens and the related P. intermedia varies among strains. For example, different strains of P. nigrescens and P. intermedia vary in their ability to adhere to buccal epithelial cells (25), invade human oral epithelial cells (11), agglutinate mammalian erythrocytes, produce enzymes which include lecithinase (14), elastolytic serine protease (33) or acid phosphatases (9), and degrade IgG (22). In this communication, we report the identification of an exopolysaccharide that was unique to a clinical strain of P. nigrescens isolated from an oral lesion.…”

Kinetic analysis of PPi-dependent phosphofructokinase fromPorphyromonas gingivalis

“…The open reading frame (ORF) was subcloned into the T7‐based bacterial expression plasmid pET21a (Novagen) as previously described [8]. The PCR product was restricted and cloned into the same sites of pET21a as previously described [9]. The resulting clone, pET21a‐PgPFK, was confirmed by DNA sequencing and introduced into E. coli BL21(DE3)/pLysS.…”