The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic

Biochemical and Biophysical Research Communications 397 (2010) 745–749

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic Aaron J. Oakley * CSIRO Molecular and Health Technology, 343 Royal Parade, Parkville, Victoria 3052, Australia

a r t i c l e

i n f o

Article history: Received 25 May 2010 Available online 10 June 2010 Keywords: 3-Phytase Phytate PhyA Michaelis complex Aspergillus niger Protein structure

a b s t r a c t Phytases hydrolyse the phosphomonoesters of phytate (myo-inositol-1,2,3,4,5,6-hexakis phosphate) and thus find uses in plant and animal production through the mobilisation of phosphorus from this source. The structure of partially deglycosylated Aspergillus niger PhyA is presented in apo form and in complex with the potent inhibitor myo-inositol-1,2,3,4,5,6-hexakis sulfate, which by analogy with phytate provides a snapshot of the Michaelis complex. The structure explains the enzyme’s preference for the 30 -phosphate of phytate. The apo-and inhibitor-bound forms are similar and no induced–fit mechanism operates. Furthermore the enzyme structure is apparently unaffected by the presence of glycosides on the surface. The new structures of A. niger PhyA are discussed in the context of protein engineering studies aimed at modulating pH preference and stability. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Phytate (myo-inositol-hexakis phosphate, IHP) is the principal form of stored phosphate in plant seeds. Furthermore, inositol phosphates are a major form of organophosphorus in soil. Phytases (myo-inositol-hexaphosphate phosphohydrolases) have been identified that hydrolyse the phosphomonoester groups of IHP with a preference for the 3-(EC 3.1.3.8), 5-(EC 3.1.3.72), and 6-(EC 3.1.3.26) position on the myo-inositol ring. These enzymes sequentially dephosphorylate lower order inositol-phosphates to inositolmonophosphates. Extracellular phytases are generally lacking in plants and animals, but are commonly isolated from bacteria and fungi. Phytases are of interest to primary producers: 3-phytase from Aspergillus niger, sold as Natuphos™ (BASF animal nutrition) is added to feedstock to improve phosphorus uptake in monogastric animals that are otherwise unable to digest phytate [1]. Transgenic plants that excrete phytases into soil are being investigated with a view to increasing the inorganic phosphate available for absorption [2]. The structure and mechanism of phytases are therefore of interest. Several families of phytases are known, but the histidine acid phosphatase group represented here is the most widely studied. Structures of several 3-phytases have been determined: PhyA from A. niger [3], PhyA from Aspergillus fumigatus [4,5], PhytDc from Debaryomyces castellii CBS 2923 [6] and AppA from Escherichia coli [4]. The fold is similar in all cases: an a/b-domain similar

Abbreviations: IHP, myo-inositol hexakis phosphate; IHS, myo-inositol hexakis sulfate; RMSD, root mean square deviation; PNGase F, peptide-N-glycosidase F. * Present address: School of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia. Fax: +61 2 4221 4287. E-mail address: [email protected] 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.024

to that of the acid phosphatases and an a-domain peculiar to this family of proteins. This family possesses a conserved active site motif –RH(G/N)XRXP – for binding and catalysis and a downstream HD motif for substrate binding/product leaving [3,7]. Members of this family hydrolyse phosphomonoesters in two separate steps the formation of a phosphohistidine intermediate through a nucleophilic attack on the phosphomonoester is followed by the hydrolysis of the covalent phosphamide intermediate. An aspartate residue (in the HD motif) protonates the bridging oxygen atom to form the inositol-hydroxyl upon phosphamide formation. Several lines of evidence suggest that the histidine of the active site motif (H59 in A. niger PhyA) acts as a nucleophile and forms a covalent link with the leaving phosphate group. A phosphohistidine intermediate has been observed in the crystal structure of A. fumigatus phytase [4]. Mutation of the equivalent residue in E. coli phytase (H17A) leads to complete inactivation of the enzyme. The structure of the H17A mutant in complex with IHP shows the substrate bound with the leaving phosphate group positioned in proximity to the alanine residue introduced by the mutation [8]. To date no structures of eukaryotic 3-phytases have been determined in the presence of substrate or substrate analogue. I have therefore sought to determine the structure of A. niger phyA in complex with myo-inositol-1,2,3,4,5,6-hexakis sulfate (IHS), a potent inhibitor of this enzyme. The Ki of inhibition for phyA was estimated to be 4.6 lM [9]. Furthermore, IHS is isosteric and isoelectronic with respect to IHP, making it an excellent substrate analogue. 2. Materials and methods Kostrewa and co-workers [3] used PNGase F to fully deglycosylate PhyA prior to crystallization. Attempts to reproduce their

746

A.J. Oakley / Biochemical and Biophysical Research Communications 397 (2010) 745–749

crystals failed, and therefore a new method was developed, using endoglycosidase F1, to remove the surface glycosides from the protein prior to crystallization. NatuphosÒ 10,000 G (BASF animal nutrition) was a gift of BASF Australia Ltd. (Wyong, NSW). Natuphos granules (1 g) were added to 10 ml of buffer A (50 mM sodium chloride, 20 mM sodium acetate pH 5.4). The suspension was agitated overnight at 4 °C. Insoluble material was pelleted using an Eppendorf 5810-R centrifuge for 10 min at 4000 rpm and 4 °C. The supernatant was decanted and progressively passed through 5 lM, 0.45 lM and 0.22 lM filters to remove any remaining particles. The solution was diluted to 20 ml with buffer A to dilute salts and other low MW additives present in the Natuphos granules and concentrated to 10 ml (11 mg/ml protein) using Amicon 10 K MWCO concentrators (Millipore). To 1 ml of this solution, 25 ll of endoglycosidase F1-GST fusion protein (0.8 mg/ml) was added. Endoglycosidase F1-GST was prepared as described previously [10]. This mixture was incubated for 24 h at 37 °C and then subjected to gel filtration using a Biologic FPLC (Bio-Rad) equipped with a Superdex-200 26/60 column (GE Healthcare). The sample was passed through the column at a flow-rate of 4 ml/min using buffer B (50 mM Tris HCl, 150 mM sodium chloride, 0.02% w/v sodium azide pH 8.5). The protein appeared as a single peak with molecular weight corresponding to a monomer, which was collected and concentrated to 13 mg/ml for crystallization trials. Deglycosylation was confirmed by a shift of molecular weight on SDS–PAGE from 60 to 47 kDa. All crystals were grown at the Collaborative Crystallization Center (http://www.csiro.au/c3) using the vapour diffusion method. Screens were conducted using the JCSG + screen [11]. 50 ll of screening solution was dispensed into the reservoirs of Innovadyne plates, and equal volumes of phytase were mixed with the precipitants (total volume of 400 nl). The trays were incubated in and drops imaged by Rigaku Minstrel systems at 8 or 20 °C. Crystals for X-ray data collection were grown using GreinerÒ 24-well

plates. The vapour–diffusion hanging drop technique was used. Reservoirs contained 1 ml of PEG 3350 (15–30% w/v) and ammonium nitrate (0.1–0.4 M). Siliconized coverslips (Hampton Research) were used to suspend a drop containing 2 ll of phytase (13 mg/ml) and 2 ll of reservoir solution over the reservoir. X-ray data were collected at the Australian Synchrotron, beamline MX-2 using Blu-Ice [12]. Crystals were cryo-protected by soaking in artificial mother liquor (30% PEG 3350, 50 mM ammonium nitrate) for 30 min prior flash cooling at 100 K. A complex with IHS was prepared by directly seeding crystals of the solid compound into drops 24 h prior to X-ray analysis. The structure was solved by molecular replacement in MOLREP [13] using the previously published structure (PDB: 1IHP) with all non-protein entities removed as the search model. Electron density inspection and model building was performed using COOT [14]. Structure refinement was performed in REFMAC5 [15,16]. Maximum likelihood weights from REFMAC5 were used to calculate electron density maps with 2mFo-DFc and mFo-DFc coefficients. The new PhyA structure was used as a template to search for homologuous proteins using DALI [17]. LSQMAN [18] was used to compute pairwise RMSDs between representative PhyA homologues. A structure-based sequence alignment was created using STAMP [19] with mark-up performed by ALINE [20]. Electrostatic potential surfaces were calculated by COOT [14]. 3. Results Crystals grew as clusters of thin plates, appearing within 13 days. The crystals were of different symmetry (P21) and packing compared to the previously reported structure (P321). The structure solution was trivial, with two solutions found automatically corresponding to two monomers in the asymmetric unit. All water molecules, glycosides and ligands observed in electron density were included in models during the building phase. Inspection of

Table 1 X-ray data and refinement statistics.

a

Structure

Native

IHS complex

PDB ID

3K4P

3K4Q

X-ray data: Space group Unit cell (Å,°) Resolution range (Å) Total number of observations Number of unique reflections I/rIb R-mergec (%) Completeness Multiplicity hBi from Wilson plot (Å2)

P21 a = 71.066, b = 87.5, c = 82.2, b = 110.98 87–2.40 (2.53–2.40)a 123,184 (18,899) 35,613 (5338) 6.6 (2.4) 13.8 (54.2) 96.6 (99.8) 3.5 (3.5) 35.4

P21 a = 70.7, b = 87.6, c = 81.8, b = 110.6 76–2.20 (2.32–2.20) 173,108 (25,430) 46,790 (6741) 8.8 (2.4) 11.0 (44.2) 98.5 (95.9) 3.7 (3.8) 26.0

Refinement statistics: Resolution range (Å) Number of reflections used in refinement Number of reflections (R-free set) R-workd (%) R-freed (%) Number of atoms hBi of structure (Å2)

66–2.40 (2.46–2.40) 33,827 (2683) 1770 (124) 21.8 (24.9) 27.5 (35.3) 7195 26.97

62–2.20 (2.26–2.20) 44,411 (3403) 2377 (185) 19.7 (26.3) 25.4 (33.5) 7350 27.15

RMS deviations from ideal geometry: Bond lengths (Å) Bond angles (°) Chiral-centres (Å3) General planes (Å)

0.016 1.68 0.111 0.008

0.019 1.86 0.125 0.009

Numbers in parentheses refer to the highest resolution bin. Numbers in angular parentheses refer to mean values. R-merge = RhRi |Ihi  hIh i |/RhRiIhi. d R-factor = Rh ||Fobs|  |Fcalc||/R|Fobs| where Fobs and Fcalc are the respectively the observed and calculated structure factors. R-free was calculated from 5% of the diffraction data not used in refinement. b

c

747

A.J. Oakley / Biochemical and Biophysical Research Communications 397 (2010) 745–749

the mFo-DFc electron density map of the data from IHS-soaked crystals revealed significant (>6r) peaks in the vicinity of the active site. This was easily interpreted as IHS. X-ray data and structure refinement statistics are given in Table 1. The structure consists of an a/b-domain domain, an a-domain and an N-terminal extension (Fig. 1). N-acetylglucosamine residues were observed attached at four sites (N82, N184, N316 and N353) out of a theoretically possible eight. The fully deglycosylated PhyA (PDB: 1IHP) is similar to the partially deglycosylated enzyme reported here: they superimpose with a RMSD of 0.505 Å over 434 Ca atoms. Apart from minor shifts in the orientation of the N-acetylglucosamine-bearing asparagine residues, there are few differences between the structures. Some movement in the side-chains of E78 and T228 occurred to accommodate the N-acetylglucosamine residue bound to N82. The active site lies in a cavity formed by helices a, e, f, h, j and l (Fig. 1). The catalytic histidine lies in a loop between strand A and helix a. Residues that are involved in IHS binding are found scattered across both domains, but the majority are in the a/b-domain. Active-site residues contact IHS through all sulfate-groups except the 60 -sulfate, which is oriented toward the solvent (Fig. 2A). There are no direct interactions of the enzyme with the inositol ring. The apo- and IHS-bound forms of phyA are very similar, superimposing with a RMSD of 0.244 Å. Binding of IHS results in displacement of a water molecule adjacent to H59 but no significant movement of the active-site residues involved in inhibitor binding. The 10 , 30 , 40 , 50 and 60 -sulfate groups of IHS-bound to PhyA are in the equatorial conformation and the 20 -sulfate group in the axial position. A crucial interaction was observed between the catalytic nucleophile H59 and the 30 -sulfate group. The H59-Ne2 atom is 3.3 Å from the S atom and is aligned with the S–O bond linking the sulfate group to inositol, with the 3 atoms describing an angle of 176°. This sulfate-group also has the lowest B-factors of all 6 and is the most deeply buried in the active-site pocket. In addition, R58, R62, R142 and D339 interact with this group through hydrogen bonds and salt bridges (Fig. 2A). Several structural homologues of PhyA were found by the DALI search, including A. fumigatus PhyA, D. castellii CBS 2923 PhytDc, A. niger pH 2.5 phosphatase, Human and Rat acid phosphatases, E. coli acid glucose-1-phosphatase (Agp) and E. coli 3-phytase (AppA). The STAMP alignment (Fig. S1) shows that the greatest conservation

Fig. 1. Ribbon diagram of A. niger PhyA with IHS (yellow) bound. The side-chains of cysteine residues involved in disulfide bonds are shown as spheres. The domains are coloured and secondary-structure elements labelled as described previously [3] (strands are uppercase letters, helices lowercase letters). The a/b domain is in red, the all-a domain is in blue, and the N-terminal leader sequence is green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

A

Y28 Q27

5

4

R62

D339

R58

T65 6

R58

4

H59

H250

R20

R142

6

3

H17A R92

D90

2 H303

1

R267

4

R20

T23

D304

R16

H250

M216

E219

K24

5

4

1

2 H338

D188

M216

E219

6

3

D188

B

K68 5 T65

D339

1

2 H338

R142

K278

Q27 R62

3 H59

Y28

K68

K278

K24

5 T23

D304

R16 H17A R92

6

3

2 1 H303

R267

D90

Fig. 2. The active-site residues of PhyA (A) and E. coli AppA; (B) ligands are drawn with black carbon atoms. The scissile bond is indicated by the scissors symbol. Each substituent is numbered.

between the structures lie in their a/b-domains, and that the a-domains show the greatest variability (Fig. S1). The A. fumigatus PhyA shares the most similarity with the A. niger enzyme (Table 2). The residues in direct contact with IHS in A. niger PhyA are absolutely conserved in A. fumigatus PhyA. 4. Discussion Successful engineering of enzymes for desirable catalytic properties is assisted by availability of structures in catalytically relevant states. Several lines of evidence suggest that IHS is bound to PhyA in manner similar to IHP and that the PhyA/IHS structure is an excellent proxy for the Michaelis complex. Consistent with functional group preference, the inhibitor is bound with 30 -sulfate adjacent to the catalytic histidine. The structure suggests an alignment of the histidine nucleophile with the 30 -phosphate that is optimal for nucleophilic attack. Residue D339 lies adjacent to the scissile bond and, consistent with the proposed mechanism, is most likely protonated on the Od2-atom and donates a hydrogen bond to the oxygen atom bridging the 30 -sulfate group and the inositol ring. In the proposed mechanism, the hydrogen would be transferred to the newly formed inositol-hydroxyl upon formation of the covalent phosphamide intermediate. The similarity of the apo- and IHS-bound forms of A. niger PhyA shows that an induced–fit mechanism does not operate. By contrast, IHP binding in E. coli AppA results in significant movements in the phytatebinding residues R20, T23 and K24 (Fig. 2B) [8]. The binding of IHS to A. niger phytase provides a structural basis for the preference of this enzyme for 30 -phosphate groups of IHP. The inositol ring of IHS is oriented with all substituents except the 20 -sulfate in an equatorial conformation (Fig. 2A). The exceptional nature of the 20 -group of IHS appears to be the basis for orientation of the molecule in the active site. The binding of the

748

A.J. Oakley / Biochemical and Biophysical Research Communications 397 (2010) 745–749

Table 2 Pairwise similarity of representative PhyA homologues. 1DKQ PhyA 1QWO 2GFI 1QFX 1ND6 1RPT 1NT4 1DKQ

1.734 1.559 2.087 1.537 1.616 1.629 1.371 410

1NT4 (208) (199) (100) (141) (230) (207) (295)

1.780 1.591 1.719 1.753 1.562 1.565 391

1RPT (195) (150) (141) (192) (191) (179)

1.369 2.156 1.261 1.228 0.815 342

1ND6 (175) (120) (134) (123) (338)

1.442 2.075 1.488 1.313 354

(191) (111) (197) (170)

1QFX

2GFI

1QWO

PhyA

1.589 (215) 1.625 (288) 1.274 (394) 460

1.540 (300) 2.164 (122) 458

0.622 (417) 442

438

RMSD between pairs of structures in Å. Parentheses contain the number of Ca’s used in the comparison. On the diagonal in bold are the total number of Ca’s in each structure. Structures are: 1QWO (A. fumigatus PhyA), 2GFI (D. castellii PhytDc), 1QFX (A. niger pH 2.5 acid phosphatase), 1ND6 (human prostatic acid phosphatase), 1RPT (Rat acid phosphatase), 1NT4 (E. coli periplasmic glucose-1-phosphatase) and 1DKQ (E. coli AppA).

20 - sulfate group occurs in a sub-site specific for an axial substituent: adjacent to the face of the imidazole ring of H338. An axially oriented sulfate group would be less energetically favourable in any other sub-site, as binding would require distortion of the inositol ring away from the chair conformation. Analogous binding is observed in the E. coli AppA/IHP complex (Fig. 2B). It is noteworthy that an inverted IHS conformer occurs in the complex with Selenomonas ruminantium 5-phytase (PDB ID: 1U26) [21]: all groups are axial except for the equatorial 20 -substituent. The 5-phytase binds the axial 10 , 40 50 and 60 - sulfate groups. The conformation that is most stable in solution appears to be dependant on pH and the type and concentration of counter-ions, however the two forms appear readily inter-convertible [22]. It is noteworthy that the structures of partially deglycosylated PhyA presented here are practically identical to the fully deglycosylated enzyme structure reported previously [3]. It was reported that PNGase F treated PhyA lost 30% of its activity [10]. Endo F1treatment used in this study leaves a single N-acetyl glucosamine group attached on asparagine residues, whereas the PNGase F used previously removes all carbohydrate groups. We conclude that the loss of activity stems neither from structural perturbations or participation of carbohydrate moieties in catalysis, but from decreased stability of the protein. A. niger PhyA has an unusual pH–Activity profile, with optima at pH 2.5 (60%) and 5–5.5 (100%) with a local minimum at pH 3.5 [23]. In contrast, A. fumigatus PhyA has one optimum at pH 5.5 to 6.5 [5]. The crystals used here were grown at a relatively high pH 8.5, but the conformation of active-site residues are the same in the structure at pH 6.5 determined by Kostrewa and co-workers [3]. In contrast, the apo-enzyme structure of A. fumigatus phytase displays pH dependence: the structure of the apoenzyme at pH 5.5, at which it is most active, differs slightly from the apo-enzyme structure at pH 7.5 through movement of H338 away from the 30 phosphate binding site [5]. The structure of A. fumigatus PhyA with phosphate bound adjacent to H59 (at both pH 5.5 and 7.5) resembles the apoenzyme at pH 7.5. The lower activity at pH 7.5 was attributed of three tightly bound water molecules at positions equivalent to three of the oxygen atoms of the substrate/reaction product 30 -phosphate group. The water molecules mimic the substrate/product and prevent substrate binding. In the apo-enzyme structure determined here, only one water molecule was observed in the 30 -phosphate binding site, 3.1 Å from the H59Ne2 atom. Comparison of PhyA with structurally conserved homologues provides insight into key regions for binding and catalysis (Fig. S1). The a/b-domain, and in particular the core b-sheet is well conserved among homologues. The position of catalytic residues found in the a/b-domain, is highly similar. Some structural and topological differences occur in the a-domains. An examination of the a-domains in the various homologues shows that while few residues are involved in substrate binding, there is a substantial contribution to the formation of the rim of the binding cavity.

In PhyA, the a-domain appears to be involved in the stabilization of residues that contact the 40 - and 50 -sulfate groups. E coli proteins Agp and Appa have a two-stranded beta sheet in place of helix e and f (Fig. 1) that forms the rim of the cleft in which the active site lies. Acknowledgments I thank the staff of the C3 centre for assistance with crystallization. I thank William McKinstry and Christine Robinson for preparing the endoglycosidase F1. I thank Alan Richardson for materials and helpful discussions. This research was undertaken on the MX2 beamline at the Australian Synchrotron, Victoria, Australia. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.06.024. References [1] R.J. Wodzinski, A.H.J. Ullah, Phytase, Advances in Applied Microbiology 42 (42) (1996) 263–302. [2] T.S. George, R.J. Simpson, P.A. Hadobas, A.E. Richardson, Expression of a fungal phytase gene in Nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils, Plant Biotechnology Journal 3 (2005) 129–140. [3] D. Kostrewa, F.G. Leitch, A. DArcy, C. Broger, D. Mitchell, A.P.G.M. vanLoon, Crystal structure of phytase from Aspergillus ficuum at 2.5 angstrom resolution, Nature Structural Biology 4 (1997) 185–190. [4] T. Xiang, Q. Liu, A.M. Deacon, M. Koshy, I.A. Kriksunov, X.G. Lei, Q. Hao, D.J. Thiel, Crystal structure of a heat-resilient phytase from Aspergillus fumigatus, carrying a phosphorylated histidine, Journal of Molecular Biology 339 (2004) 437–445. [5] O. Liu, Q.Q. Huang, X.G. Lei, Q. Hao, Crystallographic snapshots of Aspergillus fumigatus phytase, revealing its enzymatic dynamics, Structure 12 (2004) 1575–1583. [6] M. Ragon, F. Hoh, A. Aumelas, L. Chiche, G. Moulin, H. Boze, Structure of Debaryomyces castellii CBS 2923 phytase, Acta Crystallographica, Section FStructural Biology and Crystallization Communications 65 (2009) 321–326. [7] D.C. Lee, M.A. Cottrill, C.W. Forsberg, Z.C. Jia, Functional insights revealed by the crystal structures of Escherichia coli glucose-1-phosphatase, Journal of Biological Chemistry 278 (2003) 31412–31418. [8] D. Lim, S. Golovan, C.W. Forsberg, Z.C. Jia, Crystal structures of Escherichia coli phytase and its complex with phytate, Nature Structural Biology 7 (2000) 108–113. [9] A.H.J. Ullah, K. Sethumadhavan, Myo-inositol hexasulfate is a potent inhibitor of Aspergillus ficuum phytase, Biochemical and Biophysical Research Communications 251 (1998) 260–263. [10] F. GrueningerLeitch, A. DArcy, B. DArcy, C. Chene, Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases, Protein Science 5 (1996) 2617–2622. [11] J. Newman, D. Egan, T.S. Walter, R. Meged, I. Berry, M. Ben Jelloul, J.L. Sussman, D.I. Stuart, A. Perrakis, Towards rationalization of crystallization screening for small- to medium-sized academic laboratories: the PACT/JCSG plus strategy, Acta Crystallographica, Section D-Biological Crystallography 61 (2005) 1426– 1431. [12] T.M. McPhillips, S.E. McPhillips, H.J. Chiu, A.E. Cohen, A.M. Deacon, P.J. Ellis, E. Garman, A. Gonzalez, N.K. Sauter, R.P. Phizackerley, S.M. Soltis, P. Kuhn, Blu-Ice and the Distributed Control System: software for data acquisition and instrument control at macromolecular crystallography beamlines, Journal of Synchrotron Radiation 9 (2002) 401–406.

A.J. Oakley / Biochemical and Biophysical Research Communications 397 (2010) 745–749 [13] A.A. Lebedev, A.A. Vagin, G.N. Murshudov, Model preparation in MOLREP and examples of model improvement using X-ray data, Acta Crystallographica, Section D: Biological Crystallography 64 (2008) 33–39. [14] P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics, Acta Crystallographica, Section D: Biological Crystallography 60 (2004) 2126–2132. [15] G.N. Murshudov, A.A. Vagin, E.J. Dodson, Refinement of macromolecular structures by the maximum-likelihood method, Acta Crystallographica, Section D-Biological Crystallography 53 (1997) 240–255. [16] G.N. Murshudov, A.A. Vagin, A. Lebedev, K.S. Wilson, E.J. Dodson, Efficient anisotropic refinement of macromolecular structures using FFT, Acta Crystallographica, Section D-Biological Crystallography 55 (1999) 247–255. [17] L. Holm, C. Sander, Protein structure comparison by alignment of distance matrices, Journal of Molecular Biology 233 (1993) 123–138. [18] G.J. Kleywegt, Use of non-crystallographic symmetry in protein structure refinement, Acta Crystallographica, Section D: Biological Crystallography 52 (1996) 842–857.

749

[19] R.B. Russell, G.J. Barton, Multiple protein sequence alignment from tertiary structure comparison: assignment of global and residue confidence levels, Proteins 14 (1992) 309–323. [20] C.S. Bond, A.W. Schuttelkopf, ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments, Acta Crystallographica, Section D: Biological Crystallography 65 (2009) 510–512. [21] H.M. Chu, R.T. Guo, T.W. Lin, C.C. Chou, H.L. Shr, H.L. Lai, T.Y. Tang, K.J. Cheng, B.L. Selinger, A.H.J. Wang, Structures of Selenomonas ruminantium phytase in complex with persulfated phytate: DSP phytase fold and mechanism for sequential substrate hydrolysis, Structure 12 (2004) 2015–2024. [22] A.J. Costello, T. Glonek, T.C. Myers, 31P nuclear magnetic resonance-pH titrations of myo-inositol hexaphosphate, Carbohydrate Research 46 (1976) 159–171. [23] E.J. Mullaney, C.B. Daly, T. Kim, J.M. Porres, X.G. Lei, K. Sethumadhavan, A.H. Ullah, Site-directed mutagenesis of Aspergillus niger NRRL 3135 phytase at residue 300 to enhance catalysis at pH 4.0, Biochemistry Biophysics Research Communication 297 (2002) 1016–1020.