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, Mn 2؉ is also found in the enzyme. A third metal ion, Ca 2؉ , 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 Mn 2؉ 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 Ca 2؉ . 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.Most phospholipase D (PLD, 1 EC 3.1.4.4) enzymes known to date belong to a superfamily whose hallmark is an HKD motif that is essential for catalyzing the hydrolysis of phospholipids to phosphatidic acid and the corresponding free base (1, 2). The PLD superfamily includes enzymes of bacterial, plant (3, 4), and mammalian (5) origin. Whereas the bacterial enzymes are secreted and are believed to be involved in host infectivity and phosphate retrieval, the eukaryotic counterparts are involved in intracellular signal transduction through the generation of the bioactive molecule phosphatidic acid (PA). PLDs in this family also share a two-step hydrolytic mechanism involving nucleophilic attack by histidine on the phosphodiester with formation of a phosphatidylhistidine intermediate accompanied by release of a base, followed by attack of water (or an alcohol) on the phosphohistidine intermediate to release PA (or the transphosphatidylation product (6 -8)) and regenerate free enzyme (2, 9 -11). Despite the large effort expended in the characterization of the physiological role of PLD enzymes, it still remains to be determined to what extent the members of the PLD superfamily are involved in signal transduction and whether the production of the second messenger PA can occur by other than an HKD-PLD.Phospholipase D from Streptomyces chromofuscus (scPLD) while clearly able to generate PA from phospholipids, does not contain an HKD motif (12). Furthermore, scPLD requires a much higher water-soluble alcohol concentration (8 -10 M) to reach maximum transferase activity (13). Almost no transphosphatidylation activi...

Crystal Structure of the Bacillus subtilis Phosphodiesterase PhoD Reveals an Iron and Calcium-containing Active Site

“…Within this structural superfamily it has been argued that the PhoD family has greatest similarity to eukaryotic purple acid phosphatases (PAPs) (9). In PAPs the active site metal pair are a Fe 3+ ion and a divalent ion that can be Fe 2+ , Mn 2+ , or Zn 2+ depending on the enzyme (10, 11).…”

BacillussubtilisPhosphodiesterasePhoD

“…The PAPs hydrolyze a broad range of substrates but it is unknown whether their function is promiscuous -as the general alkaline phosphatases PhoA and PhoX are thought to be, or with specific function, as in the PhoD homologue S. chromofuscus PLD, which is thought to have a dedicated role removing the polar head from phospholipid substrates. On the basis of sequence alignment, PhoD was predicted to bear resemblance the PAPs, 14 and indeed, upon structural solution of PhoD, this was found to be the case.…”

Characterisitics and Gene Cloning of Phospholipase D of the Psychrophile,Shewanellasp.

“…As shown in Table 2 the active center, and the Ca 2þ ion is involved in binding to the substrate. 7) Complete inhibition of swPLD by the Fe 3þ and Mn 2þ ions suggests that the structure of the active center was different from that of scPLD. Enhancement of transphosphatidylation with the Ca 2þ ion was rather higher than the hydrolysis, suggesting that the Ca 2þ ion might be important to the action of the OH À ion and to recognition of alcohol as an acceptor of the phosphatidyl group.…”

“…5,6) As to S. chromofuscus PLD, it has been suggested that the enzyme requires Fe 3þ and Mn 2þ ions, in addition to the Ca 2þ ion, for construction of the catalytic site. 7) On one hand, these bacterial secretion types of PLDs might generate organophosphates for phosphate retrieval in media, and PLDs, in some bacteria, are important for their physiology as virulence determinants. 8,9) However, with respect to the enzymatical properties and the physiological function of bacterial PLDs, little is known.…”