title: Mutagenesis of putative catalytic and regulatory residues of Streptomyces chromofuscus phospholipase D differentially modifies phosphatase and phosphodiesterase activities.
abstract: Phospholipase D from Streptomyces chromofuscus (sc-PLD) is a member of the diverse family of metallo-phosphodiesterase/phosphatase enzymes that also includes purple acid phosphatases, protein phosphatases, and nucleotide phosphodiesterases. Whereas iron is an essential cofactor for scPLD activity, Mn2+ is also found in the enzyme. A third metal ion, Ca2+, has been shown to enhance scPLD catalytic activity although it is not an essential cofactor. Sequence alignment of scPLD with known phosphodiesterases and phosphatases requiring metal ions suggested that His-212, Glu-213, and Asp-389 could be involved in Mn2+ binding. H212A, E213A, and D389A were prepared to test this hypothesis. These three mutant enzymes and wild type scPLD show similar metal content but considerably different catalytic properties, suggesting different roles for each residue. His-212 appears involved in binding the phosphate group of substrates, whereas Glu-213 acts as a ligand for Ca2+. D389A showed a greatly reduced phosphodiesterase activity but almost unaltered ability to hydrolyze the phosphate group in p-nitrophenyl phosphate suggesting it had a critical role in aligning groups at the active site to control phosphodiesterase versus phosphatase activities. We propose a model for substrate and cofactor binding to the catalytic site of scPLD based on these results and on sequence alignment to purple acid phosphatases of known structure.
authors.affiliation: Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
authors.forename: Carlo
authors.initials: C
authors.lastname: Zambonelli
authors.forename: Monica
authors.initials: M
authors.lastname: Casali
authors.forename: Mary F
authors.initials: MF
authors.lastname: Roberts
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002151:Calorimetry; D020134:Catalytic Domain; D002942:Circular Dichroism; D005973:Glutamine / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D007477:Ions; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D010739:Phospholipase D / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology*; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D006023:Glycoproteins; D007477:Ions; D005973:Glutamine; D001224:Aspartic Acid; D006639:Histidine; D007501:Iron; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases; D010727:Phosphoric Diester Hydrolases; D010739:Phospholipase D; D008274:Magnesium; D002118:Calcium