Purple acid phosphatases (PAP) contain a dinuclear Fe(III)/Me(II) center (Me = Fe, Zn or Mn) and catalyze the hydrolysis of activated phosphoric acid monoesters in a pH range from 4 to 7. The characteristic purple colour results from a tyrosinate -> Fe(III) charge transfer transition at about 560 nm. PAPs were isolated from plant, mammalian and fungal sources. They differ from metal-independent acid phosphatases in their insensitivity to tartrate inhibition.
Due to the widespread occurence and the low substrate specifity of these enzymes functional aspects remain partly unclear. Possible biological functions of this class of enzymes include iron-transport, hydrolysis and redox reactions. PAP from red kidney bean (kbPAP) was the first PAP for which an X-ray structure was determined and which is best characterized so far.
In a one step reaction mechanism of phosphoric acid ester hydrolysis by PAPs deduced from crystal structure analysis interactions of two histidines with the substrate (His92 and His195 for Uf, His202 and His296 for kbPAP) are presumed. By electrostatic interaction the substrate correctly orientates for nucleophilic attack and becomes more electrophilic. In addition His195 (His296) is thought to act as general acid-base catalyst by protonating the leaving alcohol. In kbPAP an additional histidine (His295) comes into close contact to the substrate and might regulate substrate specifity. For investigation of these histidines kbPAP mutants encoding a single amino acid exchange at position His202, 295 or 296 are recombinantly expressed and will be characterized by kinetic and structural analysis.

The two metalloproteins catechol oxidase (Ipomoea batatas) and tyrosinase (Agaricus bisporus) are also subject of our research. Catechol oxidase and tyrosinase as well as hemocyanin belong to a group of enzymes with dinuclear copper type III active sites. Contrary to the structural homology of their active sites, they differ in catalytic properties. We want to learn how structure controls the protein function (protein crystalography) and upon this design proteins ("genetic engineering") with more specific functionality, which could be used for bio-sensors.

Research group: Dr. A. Rompel, Prof. Dr. Bernt Krebs

Publications

• N. Sträter, B. Jasper, M. Scholte, B. Krebs, A. P. Duff, D. B. Langley, R. Han, B. A. Averill, H. C. Freeman, J. M. Guss
  "Crystal Structures of Recombinant Human Purple Acid Phosphatase in the Active Form and with an Inhibitory Conformation of the Repression Loop"
  J. Mol. Biol. 2005, 351, 233.

• J. I. Naseri, N. T. Truong, J. Hörentrup, P. Kuballa, A. Vogel, A. Rompel, F. Spener, B. Krebs
  "Porcine Purple Acid Phosphatase: Heterologous Expression, Characterization and Proteolytic Analysis "
  Archives of Biochem. and Biophys. 2004, 432, 25.

• B. Jasper, N. Sträter, B. Krebs
  "Crystal Structure of the recombinant human purple acid phosphatase"
  Z. Kristallogr. (Suppl.) 2004, 21, 18

• A. Vogel, T. Böttchers, K. Markus, H. E. Meyer, B. Krebs u. F. Spener
  "Heterologous expression and characterization of recombinanat purple acid phosphatase from red kidney bean"
  Archives of Biochemistry and biophysics 2002, 401, 164

• A. Rompel, C. Gerdemann, A. Vogel u. B. Krebs
  "Enzyme structures: Active site structural and functional aspects of purple acid phosphatase and catechol oxidase"
  Buchbeitrag in : "Inorganic Chemistry Highlights" Wiley VCH-Verlag Weinheim 155, 2002

• C. Gerdemann, C. Eicken u. B. Krebs
  "The crystal structure of catechol oxidase: New insight into the function of copper type-3 proteins"
  Acc. Chem. Res. 2002, 35, 183

• C. Gerdemann, C. Eicken, H. - J. Galla u. B. Krebs
  "Comparative modelling of the latent form of a plant catechol oxidase using a molluscan hemocyanin structure"
  J. Inorg. Biochem. 2002, 89, 155

• C. Eicken, C. Gerdemann u. B. Krebs
  "Catechol Oxidase"
  in: Handbook of Metalloproteins, John Wiley & Sons, Ltd. 2001, Chichester, Vol. 2, 1319

• A. Vogel, F. Spener u. B. Krebs
  "Purple Acid Phosphatase"
  in: Handbook of Metalloproteins, John Wiley & Sons, Ltd. 2001, Chichester, Vol. 2, 752

• C. Gerdemann, C. Eicken, A. Magrini, H. E. Meyer, A. Rompel, F. Spener u. B. Krebs
  "Isozymes of Ipomea batatas catechol oxidase differ in catalase-activity"
  Biochem. Biophys. Acta 2001, 1548, 94

• B. Krebs, C. Eicken, C. Gerdemann, K. Büldt-Karentzopoulos, A. Magrini, T. Klabunde u. J. C. Sacchettini
  "Crystal structure, sequence, and mechanism of catechol oxidase"
  J. Inorg. Biochem. 1999, 74, 35

• C. Eicken, B. Krebs u. J. C. Sacchettini
  "Catechol Oxidase - Structure and Activity"
  Curr. Opin. in Struct. Biol. 1999, 9, 677

• A. Durmus, C. Eicken, F. Spener u. B. Krebs
  "Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas) "
  BBA - Protein Struct. & Mol. Enzymol. 1999, 1434, 202

• B. H. Sift, A. Durmus, W. Meyer-Klaucke u. B. Krebs
  "EXAFS Studies on the Active Site of Purple Acid Phosphatase from Sweet Potato Ipomoea batatas"
  J. Synchr. Rad. 1999, 6, 421

• A. Rompel, H. Fischer, D. Meiwes, K. Büldt-Karentzopoulos, A. Magrini, C. Eicken, C. Gerdemann u. B. Krebs
  "Substrate specificity of catechol oxidase from Lycopus europaeus and characterization of the bioproducts of enzymic caffeic acid oxidation"
  FEBS Letters 1999, 445, 103

• A. Durmus, C. Eicken, B. Sift, A. Kratel, R. Kappl, J. Hüttermann u. B. Krebs
  "The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas): Metal content and spectroscopic characterization"
  Eur. J. Biochem. 1999, 260, 709

• A. Rompel, H. Fischer, D. Meiwes, K. Büldt-Karentzopoulos, R. Dillinger, F. Tuczek, H. Witzel u. B. Krebs
  "Purification and spectroscopic studies on catechol oxidases from Lycopus europaeus and Populus nigra: Evidences for a dinuclear copper center of type 3 and spectroscopic similarities to tyrosinase and hemocyanin"
  JBIC 1999, 4, 56

• B. H. Sift, A. Durmus, W. Meyer-Klaucke u. B. Krebs
  "Photoreduction of the active sites of purple acid phosphatases by high-energy synchrotron radiation"
  EMBL Annual Report 1998, 411

• C. Eicken, F. Zippel, K. Büldt-Karentzopoulos and B. Krebs
  "Biochemical and spectroscopic characterization of catechol oxidase from sweet potatoes (Ipomoea batatas) containing a type-3 dicopper center"
  FEBS letters 1998, 486, 293

• T. Klabunde, C. Eicken, J. C. Sacchettini and B. Krebs
  "Crystal Structure of a Plant Catechol Oxidase - A Dicopper Center for Activation of Dioxygen"
  Nature Struct. Biol. 1998, 5, 1084

• H. Witzel, B. Krebs, A. Bruch, K. Büldt-Karentzopoulos, M. Dietrich, A. Durmus, C. Eicken, H. Fischer, T. Klabunde, M. Körner, D. Meiwes, D. Münstermann, R. Löcke, A. Rompel, B. Stahl, N. Sträter, H. Suerbaum
  "Phosphatase Activity and Oxygen Activation in the Active Site of the Fe(III)-Fe(II) Purple Acid Phosphatases" in: "Bioinorganic Chemistry" 1997, 385

• T. Klabunde, N. Sträter, H. Witzel u. B. Krebs
  "Violette Phosphatase: Kristallstruktur und Mechanismus"
  Z. Kristallogr. 1997, 12, 122

• T. Klabunde u. B. Krebs
  "The Dimetal Center in Purple Acid Phosphatases"
  in: " Structure and Bonding " 1997, 89, 177

• N. Sträter, W. N. Lipscomb, T. Klabunde u. B. Krebs
  "Two-Metal Ion Catalysis in Enzymatic Acyl- and Phosphoryl-Transfer Reactions"
  Angew. Chem. 1996, 108, 2158; Angew. Chem. Int. Ed. Engl. 1996, 35, 2024

• T. Klabunde, N. Sträter, R. Fröhlich, H. Witzel u. B.Krebs
  "Mechanism of Fe(III)-Zn(II) Purple Acid Phosphatase Based on Crystal Structures"
  J. Mol. Biol. 1996, 259, 737

• T. Klabunde, N. Sträter, H. Witzel u. B. Krebs
  "Purple Acid Phosphatase Containing a Dinuclear Fe(III)-Zn(II) Active Site"
  Z. Kristallogr. (Suppl.) 1995, 10, 70

• T. Klabunde, N. Sträter, H. Witzel u. B. Krebs
  "Crystal Structure of Dinuclear Fe(III)-Zn(II) Purple Acid Phosphatase"
  J. Inorg. Biochem. 1995, 59, 363

• F. Ahlers, F. Zippel, T. Klabunde, B. Krebs, R. Löcke, H. Witzel u. H.-F. Nolting
  "X-ray absorption studies of the purple acid phosphatase from red kidney beans (native enzyme, metal exchanged form)"
  Physica B 1995, 208 & 209, 731

• T. Klabunde, N. Sträter, B. Krebs u. H. Witzel
  "Structural relationship between the mammalian Fe(III)-Fe(II) and the Fe(III)-Zn(II) plant purple acid phosphatases"
  FEBS letters 1995, 367, 56

• N. Sträter, T. Klabunde, P. Tucker, H. Witzel u. B. Krebs
  "Crystal structure of dinuclear Fe(III)-Zn(II) purple acid phosphatase"
  Science 1995, 268, 1489