 |
|
|
 |
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
491 a.a.
 A phosphatidylcholine |
+ |
H(2)O |
= |
 choline |
+ |
 a phosphatidate |
|---|
Key reference
DOI no: 10.1016/j.jmb.2004.04.003 J Mol Biol 339:805-820 (2004) PubMed id: 15165852
The reaction mechanism of phospholipase D from Streptomyces sp. strain PMF. Snapshots along the reaction pathway reveal a pentacoordinate reaction intermediate and an unexpected final product. I.Leiros, S.McSweeney, E.Hough. ABSTRACT
Almost all enzyme-catalysed phosphohydrolytic or phosphoryl transfer reactions proceed through a five-coordinated phosphorus transition state. This is also true for the phospholipase D superfamily of enzymes, where the active site usually is made up of two identical sequence repeats of an HKD motif, positioned around an approximate 2-fold axis, where the histidine and lysine residues are essential for catalysis. An almost complete reaction pathway has been elucidated by a series of experiments where crystals of phospholipase D from Streptomyces sp. strain PMF (PLD(PMF)) were soaked for different times with (i) a soluble poor, short-chained phospholipid substrate and (ii) with a product. The various crystal structures were determined to a resolution of 1.35-1.75 A for the different time-steps. Both substrate and product-structures were determined in order to identify the different reaction states and to examine if the reaction actually terminated on formation of phosphatidic acid (the true product of phospholipase D action) or could proceed even further. The results presented support the theory that the phospholipase D superfamily shares a common reaction mechanism, although different family members have very different substrate preferences and perform different catalytic reactions. Results also show that the reaction proceeds via a phosphohistidine intermediate and provide unambiguous identification of a catalytic water molecule, ideally positioned for apical attack on the phosphorus and consistent with an associative in-line phosphoryl transfer reaction. In one of the experiments an apparent five-coordinate phosphorus transition state is observed.
Selected figure(s)
Figure 1.
Figure 1. The active site of PLD[PMF] unliganded or phosphate-inhibited. (a) The native state (structure 1). Two water molecules (OW1 and OW2) occupy the binding site for the phosphate moiety. (b) Phosphate-inhibited PLD[PMF] (structure 2). Both electron density maps are s[A]-weighted 2mF[o] -DF[c] maps contoured at 1.5s.Figure 8.
Figure 8. The reaction mechanism for PLD[PMF] on a phosphatidylcholine (PC) substrate. R, Diacylglycerol (DAG); R', choline. The reaction that takes place when the product re-enters the active site and the dead-end phosphohistidine is formed is illustrated below the horizontal line.The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 339, 805-820) copyright 2004. Figures were selected by an automated process.
Literature references that cite this PDB file's key reference
PubMed id Reference
19241409 C.Morasso, T.Bellini, D.Monti, M.Bassi, D.Prosperi, and S.Riva (2009).
Dispersed phantom scatterer technique reveals subtle differences in substrate recognition by phospholipase D inactive mutants.Chembiochem, 10, 639-644.
18338352 A.Masayama, T.Takahashi, K.Tsukada, S.Nishikawa, R.Takahashi, M.Adachi, K.Koga, A.Suzuki, T.Yamane, H.Nakano, and Y.Iwasaki (2008).
Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis.Chembiochem, 9, 974-981.
18398008 Z.Lu, D.Dunaway-Mariano, and K.N.Allen (2008).
The catalytic scaffold of the haloalkanoic acid dehalogenase enzyme superfamily acts as a mold for the trigonal bipyramidal transition state.Proc Natl Acad Sci U S A, 105, 5687-5692.
PDB codes: 2rar 2rav 2rb5 2rbk
17855396 S.H.Chan, Y.Bao, E.Ciszak, S.Laget, and S.Y.Xu (2007).
Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities.Nucleic Acids Res, 35, 6238-6248.
17451436 S.Stumpe, S.König, and R.Ulbrich-Hofmann (2007).
Insights into the structure of plant alpha-type phospholipase D.FEBS J, 274, 2630-2640.
17189478 Y.Uesugi, J.Arima, M.Iwabuchi, and T.Hatanaka (2007).
C-terminal loop of Streptomyces phospholipase D has multiple functional roles.Protein Sci, 16, 197-207.
17459102 Y.Uesugi, J.Arima, M.Iwabuchi, and T.Hatanaka (2007).
Sensor of phospholipids in Streptomyces phospholipase D.FEBS J, 274, 2672-2681.
16339153 L.G.Henage, J.H.Exton, and H.A.Brown (2006).
Kinetic analysis of a mammalian phospholipase D: allosteric modulation by monomeric GTPases, protein kinase C, and polyphosphoinositides.J Biol Chem, 281, 3408-3417.
15654080 M.T.Murakami, M.F.Fernandes-Pedrosa, D.V.Tambourgi, and R.K.Arni (2005).
Structural basis for metal ion coordination and the catalytic mechanism of sphingomyelinases D.J Biol Chem, 280, 13658-13664.
PDB codes: 1xx1 2f9r
15973486 R.Ulbrich-Hofmann, A.Lerchner, M.Oblozinsky, and L.Bezakova (2005).
Phospholipase D and its application in biocatalysis.Biotechnol Lett, 27, 535-544.
15899903 Y.Uesugi, K.Mori, J.Arima, M.Iwabuchi, and T.Hatanaka (2005).
Recognition of phospholipids in Streptomyces phospholipase D.J Biol Chem, 280, 26143-26151.
15691025 R.Sato, Y.Itabashi, A.Suzuki, T.Hatanaka, and A.Kuksis (2004).
Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol.Lipids, 39, 1019-1023. The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.
 |