An iron-dependent bacterial phospholipase D reminiscent of purple acid phosphatases.
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 lambda(max) at 570 nm. Metal ion analysis performed by inductively coupled plasma mass spectroscopy shows the presence of approximately 1 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.
Zambonelli
Carlo
C
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Roberts
Mary F
MF
D000135:Acid Phosphatase / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D007477:Ions; D007501:Iron / Q000494:pharmacology*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D014466:Ultraviolet Rays
D006023:Glycoproteins; D007477:Ions; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010712:Phosphatidic Acids; C008644:nitrophenylphosphate; D004492:Edetic Acid; D007501:Iron; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D010739:Phospholipase D; D002118:Calcium