The Binding Mode of an ADP Analogue to a Metallohydrolase Mimics the Likely Transition State

Abstract

Purple acid phosphatases( PAPs) are members of the large family of metallohydrolases, ag roup of enzymes that perform aw ide range of biological functions, while employing ah ighly conserved catalytic mechanism. PAPs are found in plants,a nimals and fungi;i nh umans they play an important role in bone turnovera nd are thus of interest for developingt reatments for osteoporosis. The majority of metallohydrolases use am etalbound hydroxide to initiate catalysis, which leads to the formation of ap roposed five-coordinate oxyphosphorane species in the transition state. In this work, we crystallized PAPf rom red kidney beans (rkbPAP) in the presence of both adenosine and vanadate. The in crystallo-formed vanadate analogue of ADP provides detailed insight into the binding mode of aP AP substrate, captured in as tructure that mimics the putative fivecoordinate transition state. Our observations not only provide unprecedented insightinto the mechanism of metallohydrolases, but might also guide the structure-based design of inhibitors for application in the treatment of severalhuman illnesses.Metallohydrolases form al arge familyo fm ostly bimetallic enzymes that use metal ionst oactivate both the nucleophile and scissile bond of various phosphate or amide substrates. [1][2][3][4] Examples include the antibiotic-degrading metallo-b-lactamases, pesticide-decontaminating organophosphate hydrolases, and purple acid phosphatases (PAPs). [5][6][7] PAPs, the only known metallohydrolases that require ah eterovalent Fe III M II center (with M = Fe, Zn or Mn) for catalytic activity, have been characterized from various mammals, plants and fungi. [2,5] Mostp lant PAPs use either Zn II or Mn II ,w hereas their mammalianc ounterparts employ ar edox-activei ron ( Figure 1). [8,9] The reported substrates for PAPs include adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate( ADP), phosphotyrosine and pyrophosphate. [5,10] The synthetic substrate para-nitrophenyl phosphate (pNPP) is frequently used for functionalstudies. [2,5] The biological functions of PAPs are diverse and dependent on the organism. For mammals, experiments with transgenic mice have demonstrated that PAPp lays an important role in bone metabolism. [11,12] In plants, PAPs playamajor role in the acquisition of phosphorus, especially if there is al imited supplyo fp hosphate. [13,14] Due to their diverse roles, PAPs have attracted attention either as targetsf or new treatments of osteoporosis or for applicationsi na griculture to aid nutrient uptake by crops. [11,15] PAPs operate optimally in the pH range between5 .0 and 6.5, and are characterized by ad istinct purple color due to ac harge-transfer transition between an active-site tyrosine ligand and an Fe III ion. [16,17] The crystal structures of several mammalian and plant PAPs have demonstrated that, despite the difference in the metal ion selectivity and the limited degree of sequence similarity,t heir actives ite geometries are highly conserved (Figure1). [18][19][20][21][22][23] Consequently,t he proposed mod...

Structural elements that modulate the substrate specificity of plant purple acid phosphatases: avenues for improved phosphorus acquisition in crops

“…There are two dimers (subunits A-D) in each asymmetric unit of the crystal. The overall fold of the four polypeptide chains strongly resembles those of the previously determined structures of free rkbPAP[69] and the phosphate-sulfate-and vanadate-bound complexes[18,44,46] of this enzyme (rmsd values for all Cα atoms <0.353 Å). For initial fitting of the ligands in the active site, Polder omit maps were chosen due to their ability to provide enhanced electron density when compared to simulated annealing omit maps…”

“…where the absence of electron density for the adenine moiety in the adenosine group was explained by the weak interactions holding this moiety in place [18]. For the modelled ATP complex the ribose moiety is also predicted to lack significant interactions with the enzyme, but the adenine moiety is predicted to form a hydrogen bond between an adenine nitrogen (N5) and the backbone carbonyl oxygen of Phe297.…”

“…Arabidopsis thaliana [12,16], red kidney bean [17,18], soy bean [19], tobacco [20] and tomato [16,21], as well as animal species, e.g. bovine [22], human [23,24] and pig [25,26].…”

“…The calorimetrically determined catalytic parameters (kcat = 190 s -1 and Km = 4 mM) are in good agreement with the corresponding values determined spectrophotometrically (180 s -1 and 2.6 mM, respectively). This assay can easily be adapted for other substrates of PAPs as well and will be a useful tool to establish the substrate preference of diverse PAPs.Crystallographic investigations.In a previous study we demonstrated that the soaking of rkbPAP crystals in a cryosolution containing both vanadate and adenosine leads to adenosine divanadate (ADV) being formed in a complex that mimics the transition state of ADP hydrolysis to AMP by PAP[18]. No monovanadate derivatives of adenosine (AMV) were observed.…”

Structural elements that modulate the substrate specificity of plant purple acid phosphatases: Avenues for improved phosphorus acquisition in crops

“…Docking prediction of the binding modes of (left) ADP (yellow carbons) and (right) ATP (brown carbons) to rkbPAP (green surface). The corresponding crystal structures of rkbPAP in complex with ADV (salmon carbons) (6HWR[8]) and ADPV (magenta carbons) are superimposed on the docking predictions. Metal ions are shown as orange spheres and marked with an asterisk.…”