An Iron-dependent Bacterial Phospholipase D Reminiscent of Purple Acid Phosphatases

Abstract

Recombinant phospholipase D (PLD) from Streptomyces chromofuscus (scPLD) has been characterized using colorimetric assays, spectroscopic investigations, and site-directed mutagenesis. scPLD, which shows phosphodiesterase activity toward a wide variety of phospholipids and phosphatase activity toward p-nitrophenyl phosphate, exhibits a visible absorption band with max at 570 nm. Metal ion analysis performed by inductively coupled plasma mass spectroscopy shows the presence of ϳ1 equivalent of iron, 0.27 equivalent of manganese, and 0.1 equivalent of zinc per mole of protein as isolated. The metal ion content coupled with the visible absorption feature is compatible with the presence of Fe 3؉ -tyrosinate coordination. When scPLD was dialyzed against solutions containing Mn 2؉ , Zn 2؉ or EDTA, the Fe 3؉ content was reduced to variable extents, and the residual specific activity correlated well with the residual iron content. Sequence homology with metal ion binding motifs in known alkaline phosphatases and purple acid phosphatase from red kidney bean shows that most of the residues involved in metal ion coordination are conserved among all the sequences considered. Mutation of some of these conserved residues (C123A, D151A, Y154F, and H391A) produced enzymes lacking iron with dramatically reduced PLD activity but little change in secondary structure or ability to bind to small unilamellar vesicles of phosphatidylcholine (with Ba 2؉ ) or phosphatidic acid. We suggest that scPLD is a member of a family of phosphodiesterase/ phosphatases with structural and mechanistic similarity to iron-dependent purple acid phosphatases.

Mutagenesis of Putative Catalytic and Regulatory Residues of Streptomyces chromofuscus Phospholipase D Differentially Modifies Phosphatase and Phosphodiesterase Activities

“…scPLD is also different from other known PLDs in that it contains two metal ions (iron and manganese) of which at least one (iron) is an essential cofactor for the catalytic activity (14). This scPLD reactive center is homologous to that of purple acid phosphatases (PAPs) (14) and is consistent with scPLD cleavage of phospholipids occurring by a different mechanism than HKD-type PLDs. Perhaps, enzymes belonging to the family of PAPs (15) could represent candidates for non-HKD-PLDs in cells.…”

“…Furthermore, the melting temperatures of the mutant enzymes were essentially the same as wild type scPLD (71°C). These mutants were previously shown to bind to 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles in a Ba 2ϩ dependent fashion, or to anionic SUVs in the presence of EDTA (14).…”

“…Almost no transphosphatidylation activity is detectable in the presence of 1-2 M methanol or ethanol, conditions that generate Ͼ95% transphosphatidylation product by members of the PLD superfamily (12). scPLD is also different from other known PLDs in that it contains two metal ions (iron and manganese) of which at least one (iron) is an essential cofactor for the catalytic activity (14). This scPLD reactive center is homologous to that of purple acid phosphatases (PAPs) (14) and is consistent with scPLD cleavage of phospholipids occurring by a different mechanism than HKD-type PLDs.…”

“…In AP the substitution of serine for an arginine residue important for substrate binding (R166S), produced an E. coli AP with decreased phosphatase activity but unaltered phosphodiesterase activity toward bis(pNP)P (16) indicating that the two activities could be uncoupled. In this class of metalloenzymes, mutations of residues believed to be involved in metal ion binding have diverse effects on kinetic parameters, although they have not been shown to alter the type of reactivity of the enzyme (protein phosphatase-1, an enzyme with loose but important homology with purple acid phosphatases (25) and scPLD (14), is an interesting example (26)). Of the two metal ions believed to be part of the active site of scPLD, Fe 3ϩ is essential for catalysis, whereas the second metal ion (Mn 2ϩ , Zn 2ϩ , or Fe 2ϩ ) has been postulated to be involved in the binding of substrate and/or, possibly, the release of product (14).…”

“…Previously, we showed that despite being a de facto PLD, scPLD is also able to catalyze a phosphatase reaction when p-nitrophenyl phosphate (pNPP) is used as a substrate (14). Other enzymes possess both phosphatase and phosphodiesterase activities, including alkaline phosphatase from Escherichia coli (AP) (16), nucleotide pyrophosphatases/phosphodiesterases (17), and PhoD phosphatase/phosphodiesterase from Bacillus subtilis (18,19).…”