Artificial Evolution of an Enzyme Active Site: Structural Studies of Three Highly Active Mutants of Escherichia coli Alkaline Phosphatase

doi:10.1006/jmbi.2001.5384 available online at http://www.idealibrary.com on

J. Mol. Biol. (2002) 316, 941±953

Artificial Evolution of an Enzyme Active Site: Structural Studies of Three Highly Active Mutants of Escherichia coli Alkaline Phosphatase M-H. Le Du*, C. Lamoure, B. H. Muller, O. V. Bulgakov, E. Lajeunesse A. MeÂnez and J.-C. Boulain DeÂpartement d'IngeÂnierie et d'EÂtudes des ProteÂines, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France

*Corresponding author

The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (kcat) enhancement as compare to the wildtype enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the ®rst mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium ¯uoride, which mimics the transition state. These structures reveal, in particular, that this ®rst mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium ¯uoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the ®rst phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (kcat), and explains the structural events associated with this arti®cial evolution. Keywords: activity enhancement; crystallography; mutagenesis; transition state; phosphoseryl intermediate

Introduction Present address: O. V. Bulgakov, MEEI, Harvard Medical School, Department of Ophthalmology, room 530, Berman-Gund Laboratory, 243 Charles St., Boston, MA 02180, USA Abbreviations used: AP, alkaline phosphatase; APWT, wild-type Escherichia coli alkaline phosphatase; APD153H, alkaline phosphatase mutant (Asp153!His); APD330N, alkaline phosphatase mutant (Asp330!Asn); APD153HD330N, alkaline phosphatase mutant (Asp153!His, Asp330!Asn); APD153GD330N, alkaline phosphatase mutant (Asp153!Gly, Asp330!Asn); CIP, calf intestinal alkaline phosphatase; rmsd, root-meansquare deviation. E-mail address of the corresponding author: [email protected] 0022-2836/02/040941±13 $35.00/0

Alkaline phosphatases (AP; EC 3.1.3.1) from eukaryotic origins are used extensively as colorimetric enzymes because of their high speci®c activities (kcat).1 They are 20 to 40-fold more active than the Escherichia coli enzyme,2 which is, in turn, heat-stable, and easy to produce as a free enzyme as well as genetically fused to a proteic partner. In order to take advantage of the properties of both classes of enzymes, we have mutated the active site of the E. coli enzyme, with the goal of reaching a mammalian speci®c activity.3 The E. coli AP is a serine phosphatase that catalyses the hydrolysis of phosphomonoesters with release of inorganic phosphate and alcohol.4

942 During the past decade, several studies on both the native,5,6 and mutated bacterial enzyme7 ± 11 aimed at understanding the detailed steps of the catalytic reaction of AP1,12 as well as at providing the bacterial enzyme with improved catalytic constants. E. coli AP is a dimeric metalloenzyme, each monomer comprising 449 residues, two zinc atoms at sites M1 and M2, and one magnesium atom, at site M3, in the active site.5,6 On the basis of both biochemical and structural evidence, a catalytic mechanism for the enzyme has been proposed.1,5,6,13,14 First, the substrate binds to the free enzyme to form a non-covalent complex. Then, the hydroxyl group of Ser102, activated by the zinc atom in M2, performs a nucleophilic attack at the phosphate moiety of the substrate, which leads to a covalent phosphoseryl intermediate.15 Subsequently, this intermediate is hydrolysed by a water molecule, activated by the zinc atom at M1, leading to a noncovalent enzyme-phosphate complex. Finally, the phosphate moiety is released from the complex to return to the free enzyme. Therefore, two transition states are passed through during the complete reaction, the ®rst one giving rise to the formation of the phosphoseryl intermediate, the second to the non-covalent complex. At acidic pH values, the hydrolysis of the phosphoryl-enzyme is rate-limiting, whereas at alkaline pH values it is the dissociation of the non-covalent enzyme-phosphate complex that is rate-limiting.16 The extensive studies performed on the active site of E. coli AP have demonstrated the fundamental role of the metal ions in the catalytic mechanism.5,6,13 ± 15,17 The zinc atom at M2 activates Ser102, which performs the initial attack on the phosphate group of the substrate. The zinc atom at M1 activates a water molecule, which is well positioned for the apical attack of the phosphoseryl intermediate.18 The role of the magnesium ion at the M3 site remained unclear until the recent study by Stec et al.14 showing that Ser102 is orientated in an activating conformation toward the zinc ion at M2 only when magnesium is present at M3. In addition, the octahedral geometry of the magnesium ion at M3 properly positioned a Mg-bound water molecule that plays the role of a general base in the generation of the Ser102 nucleophile, and of a general acid in the regeneration of the Ser102 hydroxyl group. The X-ray structures of various forms of bacterial and human AP have been reported.5 ± 7,12,18 ± 26 These include the structures of the wild-type and mutated E. coli AP with the third metal site being occupied by zinc or magnesium. Also, AP structures have been solved in the absence or presence of phosphate bound non-covalently or covalently to the enzyme. In particular, studies on AP mutated at position 153 revealed that Asp153 is a key residue in the stabilisation of the magnesium ion at M3. When Asp153 is mutated, the resulting enzyme needs to be activated by high magnesium concentration to reach an optimal activity.9,11 This property is of particular interest, since it is shared

Arti®cial Evolution of E. coli AP Active Site

by mammalian enzymes in which Asp153 is substituted by His. With the aim of improving the catalytic activity of the E. coli enzyme, we have used an approach based on a combination of random and directed mutagenesis, and of structural studies. The biological aspects of this study have been discussed in detail elsewhere.3 Starting from a nearly inactive mutant, we searched for compensating mutations, which were then transferred into the wild-type enzyme. One of these compensating mutations, at position 330, enhanced the catalytic activity, and gave rise to our ®rst mutant D330N (APD330N). This mutation was combined with a mutation at position 153, leading to the double point mutant D153H-D330N (APD153HD330N), and ®nally to the double point mutant D153G-D330N (APD153GD330N). Here, we detail the structural aspects of this study, and highlight how the crystal structures helped in the design of AP mutants with progressively improved properties. In addition, the description of the mutant structure at various steps along the reaction pathway partially explains the mechanism of the arti®cial evolution performed. Thus, the structure of APD330N is described in the presence of phosphate or in the presence of the transition-state analogue AlF3. The structure of APD153HD330N was determined in four different conditions. Two are presented in the absence of phosphate ion, either at low magnesium concentration and in the presence of zinc or at high magnesium concentration and in the absence of zinc. These two structures allow us to understand the magnesium activation process of the catalytic serine. Two additional structures were solved at high magnesium concentration either in the presence of phosphate or in the presence of the transition-state analogue AlF3. Finally, the structure of APD153GD330N was solved in the presence of magnesium as a free enzyme or in the presence of phosphate. The resolution of these different strucÊ and 2.6 A Ê , is compatible with tures, between 2.0 A a ®ne structure analysis.

Results Enzymatic activity The kcat and Km values for APD330N, APD153HD330N, and APD153GD330N have been determined at pH 10.0 in the absence of phosphate acceptor.3 Under these conditions, the kcat value of both double point mutant is 17-fold higher than that of APWT (Table 1), whereas the single point mutants D153H9,11 and APD330N are only fourfold and threefold more active than APWT, respectively (Table 1). The Km value of APD330N is twofold decreased, whereas it increases signi®cantly when the residue at position 153 is modi®ed. Moreover, the apparent second-order rate constants (kcat/Km) of APD330N and APD153HD330N are fourfold higher than that of APWT and APD153H (Table 1). There-

943

Arti®cial Evolution of E. coli AP Active Site Table 1. Catalytic activity of the E. coli AP mutant around positions 153 and 330 Experimental conditions

Protein

0.5 M NaCl, 10 mM MgCl2, 0.1 M Caps-NaOH pH 10

APWT

20 mM ZnCl2, 1 M diethanolamine

APD330N APaD153H APD153HD330N APD153GD330N APbD153GD330N CIPc

kcat (sÿ1)

Km (mM)

kcat/Km (10ÿ6 Mÿ1 sÿ1)

80  3

90  8

0.9

201  10 292  4 1389  110 1421  50 3202  75 3435  84

47  4 545  65 350  30 566  18 567  7 660  23

4.3 0.5 4.0 2.6 5.6 5.2

The experiments were performed at 25  C in the presence of Caps, and at 37  C in the presence of diethanolamine. From Janeway et al.11 b ‡ 10 mM MgCl2, pH 10.0. c ‡ 1 mM MgCl2, pH 9.8, from Weissig et al.27 a

fore, the mutation D330N further increases the enhanced activity due to mutation D153H as well as the overall ef®ciency of the wild-type enzyme. In addition, in the case of APD153GD330N, kcat measured in the presence of the phosphate acceptor diethanolamine reaches a value of 3202 sÿ1 (Table 1),3 of the same order as that of calf intestinal AP.27 Under these conditions, the kcat of APWT is lower than 65 sÿ1.

Determination of the crystal structures The crystals belong to the space group P6322, Ê , c ˆ 138 A Ê , as reported.25 The with a ˆ b ˆ 163 A ®rst data set collected with one crystal of D330N mutant was used to solve the structure by molecular replacement with the program AMoRe,28 using the model of the wild-type enzyme (PDB entry code 1alk). In order to observe different states of the enzyme, crystals were soaked under different conditions (Table 2). The re®nement of the eight models was performed by simulated annealing with XPLOR.29 The statistics of the data collection, molecular replacement and re®nement are shown in Table 3. The structures are all similar to that of the orthorhombic form of AP.5,6 The main differences occurred at both the N-terminal region, which is disordered in our structures, and in loop 290-294. These changes can be attributed to the difference in crystal form, as observed.25 The data indicate that the side-chain of Asn330 in all crystals occupies the same position as Asp330 in AP. The nature of the metals at the three binding sites was determined from the peak height in Fo ÿ Fc omit maps calculated at the end of the re®nement. The density observed in these maps provided information about the position and nature of the metals

Activation of the double point mutants by magnesium The relative activity of the APWT or APD330N mutant increases from 84.5 % and 82.8 % at 10ÿ5 M Mg2‡, to 100 % at 10ÿ1 M Mg2‡. In the case of APD153HD330N, it increases from 11.8 % at 10ÿ5 M Mg2‡, to 100 % at 10ÿ1 M Mg2‡, and for APD153GD330N, it increases from 25 % at 10ÿ5M Mg2‡, to 100 % at 10ÿ1 M Mg2‡ (data not shown). Therefore, the magnesium concentration does not affect the activity of APWT or APD330N signi®cantly, whereas its effect is dramatic when the mutation at position 153 is introduced.3 This result corroborates the observations made previously on APD153H.11

Table 2. Soaking conditions and crystal form Crystal form

[(NH4)2SO4] (M)

[MgCl2] (mM)

APD330N(PO4) APD330N(AlF3)

2.2

10

APD153HD330N(Zn) APD153HD330N(Mg) APD153HD330N(PO4): step 1 APD153HD330N(PO4): step 2 APD153HD330N(AlF3): step 1 APD153HD330N(AlF3): step 2

2.6 2.6 2.4 2.4 2.4 2.2

10 100 100 10 100 100

APD153GD330N(Mg) APD153GD330N(PO4): step 1 APD153GD330N(PO4): step 2

1.1 2.2 2.4

10 50 10

All soaking were performed in 50 mM Tris-HCl (pH 8.0).

Other 25 mM NaF, 0.5 mM AlCl3-6H2O 0.1 mM ZnSO4 25 mM NaH2PO4 25 mM NaF, 0.5 mM AlCl3-6H2O 20 mM ZnSO4 100 mM NaH2PO4

Soaking time 3 days 15 days 15 days 15 days 15 days 1 year 3 days 1 day 6 month 6 month

Table 3. Data collection, molecular replacement and re®nement statistics Data set

APD330N (PO4)

APD330N (AlF3)

APD153HD330N (Zn)

APD153HD330N (Mg)

APD153HD330N (PO4)

APD153HD330N (AlF3)

A. Data collection X-ray source Number of reflections Rsym (%) (last shell) I/s(I) (last shell) Ê) Resolution (A Completeness (%)

ESRF D2AM 31,594 6.2 (28.0) 26.9 (4.9) 2.4 73.1

Rigaku RUH2R 68,830 8.6 (32.1) 20.9 (3.9) 2.0 94.3

ESRF D2AM 53,663 9.04 (46.0) 24 (nd) 2.6 96.3

ESRF D2AM 33,240 7.04 (20.6) 39 (nd) 2.5 87.0

Rigaku RUH2R 36,364 11.9 (35.7) 21 (7.5) 2.5 89.2

Rigaku RUH2R 49,163 11.0 (48.0) 13.2 (2.9) 2.3 99.7

Rigaku RUH2R 43,065 13.0 (53.2) 10.2 (2.8) 2.4 99.8

LURE DWat32 32,077 11.8 (69.8) 15.6 (1.8) 2.5 83.4

B. Refinement R-factor (%) (last shell) R-free (%) (last shell) No. residues No. metallic ions No. water molecules

18.8 24.5 888 6 234

19.4 22.8 888 8 353

20.7 (47.2) 24.8 (60.7) 888 6 318

17.2 (30.6) 20.7 (32.0) 888 6 390

18.3 (25.2) 22.7 (32.3) 888 6 307

18.0 (25.7) 22.3 (28.7) 888 8 360

17.3 (24.9) 23.9 (29.4) 888 6 359

19.6 (35.1) 25.0 (38.3) 888 6 372

C.rmsd Ê) Bond length (A Bond angles (deg.) Dihedral angles (deg.) Improper angles (deg.)

0.010 1.693 24.699 1.470

0.010 1.574 24.361 1.458

0.012 1.783 25.204 1.534

0.011 1.605 24.758 1.411

0.011 1.691 24.440 1.552

0.010 1.617 24.288 1.440

0.009 1.550 24.360 1.388

0.011 1.664 24.143 1.403

D. Molecular replacement Correlation (%) R-factor (%)

72.3 31.7

nd, Not determined.

APD153GD330N (Mg) APD153GD330N (PO4)

945

Arti®cial Evolution of E. coli AP Active Site Table 4. Peak height (s) in the Fo ÿ Fc omit map after removing the metal and the ion at the product site M1 (subunit 2) APD330N(PO4) APD330N(AlF3) APD153HD330N(Zn) APD153HD330N(Mg) APD153HD330N(PO4) APD153HD330N(AlF3) APD153GD330N(Mg) APD153GD330N(PO4) a

26.0 (30.5) 36.0 (40.5) 24.5 (22.0) 31.0 (28.0) 32.0 (29.0) 34.4 (30.5) 34.5 (32.0) 11 (7)

M2 (subunit 2) 28.0 38.5 26.0 30.0 23.0 26.0 33.5 7

(28.5) (37.5) (26.5) (28.5) (26.5) (25.5) (37.0) (6)

M3 (subunit 2)

Product site (subunit 2)

16.5 (13.0) 16.5 (14.0) 24.0 (23.0) 16.0 (12.5) 4.5 ( 3.0)  3.0 ( 3.0) 16.0 (14.0) 4 (4)

16.5 (16.5) 9.0 (8.0) < 3.0 (< 3.0) < 3.0 (< 3.0) 18.5 (17.0) 13.5 (13.5) 19.5 (16.5)a 8 (8)

Ê from the position of the phosphate ion, and 5.0 A Ê from Gly153. The ion is located at 4.0 A

as well as the solvent molecules. They showed high peaks that agree with the presence of zinc at M1 and M2 sites in all structures. On the contrary, the nature of the metal at site M3 varies from one form to another, as the content of ion at the product site (Table 4). As expected, in both forms of APD153HD330N and of APD153GD330N, the mutation at position 153 destroys the hydrogen-bond network observed between Lys328-Asp153-Arg166-PO4 in AP.5,6 However, Lys328 never interacts directly with the phosphate, in contrast to what was observed in the structure of APD153H.12,23

Active site of APD330N(PO4) and APD330N(AlF3) The electronic density map calculated with the data collected from a crystal of APD330N (form APD330N(PO4)) reveals that the active site of APD330N contains a magnesium atom at M3 and a phosphate ion close to the active Ser102 (Figure 1(a)). The active site of APD330N in complex with the phosphate ion is highly conserved like that of the wild-type enzyme. In particular, Asn330 occupies the same position as Asp330. The stabilisation of the phosphate ion is similar in both enzymes concerning the residues involved in this

Figure 1. (a) and (b) Ribbon, and ball and stick representation of the active site of APD330N and of the interactions that involve residues Asp51, Ser102, Asp153, Thr155, Arg166, Glu322, Lys328, water molecules Wat1 to Wat4, and Zn1, Zn2 and Mg3. (a) In the presence of phosphate in the active site. (b) In the presence of aluminium ¯uoride in the active site. (c) Ball and stick representation in stereo view of the active site of APD330N with aluminium ¯uoride in the active site, and 1s contoured 2Fo ÿ Fc map.

946 stabilisation as well as the water molecules, as observed in the high-resolution structure of the APWT.14 When a crystal of APD330N is soaked in the presence of NaF and AlCl3 (form APD330N(AlF3)), the Fo ÿ Fc map reveals the presence of a 12s peak centred on the metal ion, compatible with the presence of AlF3. The density indicates a trigonal bipyramidal AlF3 with Ser102 Og and a water molÊ from ecule as coaxial ligands. Ser102 Og is 1.7 A the aluminium ion, and has rotated by 30  around its Ca-Ca bond as compared to the free enzyme. Ê from the aluThe apical water molecule is 1.8 A minium ion (Figure 1(b) and (c)). Furthermore, AlF3 occupies a position that is similar to that observed with the phosphate ion in APD330N(PO4). As compared to APD330N(PO4), the apical water molecule can be assimilated to the fourth oxygen atom of the phosphate group. The distances of Ê and 1.8 A Ê on either side of the aluminium 1.7 A ion are compatible with an associative transition state, as observed in the native enzyme with vanadate.30 In both APD330N(PO4) and APD330N(AlF3) active sites, the hydrogen bond network is very similar, and the number and location of the stabilising water molecules are highly conserved (Figure 1). Three water molecules, Wat1-Wat3, might play a major role in the enzymatic reaction, since they interact directly with the phosphate ion or the AlF3 molecule. Wat1 is positioned in the same region as the nucleophilic water molecule described previously.18 Wat2 bridges the phosphate ion (or AlF3) to Lys328. The Mg-bound Wat3 might play the role of a general base in the generation of the Ser102 nucleophile, and of a general acid in the regeneration of the Ser102 hydroxyl group as described.14 A fourth water molecule, Wat4, is probably important, as it interacts with Arg166, and therefore reinforces the circular network PO4(or AlF3)-Arg166-Asp153-Lys328-Wat2-PO4(or AlF3). Active site of the free APD153HD330N(Zn), and APD153HD330N(Mg) In both APD153HD330N(Zn) and APD153HD330N(Mg), Arg166 is orientated toward the solvent, and the temperature factors re¯ect the agitation of its sidechain as compared to the average temperature factor of the whole protein. This orientation is related to the absence of phosphate in the active site, which stabilises the side-chain of Arg166. Although the concentration of Zn2‡ and Mg2‡ are 0.1 mM and 10 mM, respectively, in the ®rst soaking solution of APD153HD330N (Table 2), Zn2‡ occupies the M3 site (Table 4), indicating a much higher af®nity of Zn2‡ than of Mg2‡ for this site. The magnesium ion at M3 was observed after two weeks soaking in a solution containing 100 mM magnesium and no zinc. Therefore, the differences between APD153HD330N(Zn) and APD153HD330N(Mg) illustrate the activation of the protein by the magnesium ion.

Arti®cial Evolution of E. coli AP Active Site

In APD153HD330N(Zn), the zinc ion at M3 is tetrahedral and stabilised by Asp51, Thr155, Glu322 and His153. In APD153HD330N(Mg), the magnesium is octahedral and stabilised by Asp51, Thr155 and Glu322, plus three water molecules. Ser102 interacts with Zn2 in both forms, with a distance of Ê in Ê in APD153HD330N(Zn) and 2.10 A 2.93 A APD153HD330N(Mg). However, in APD153HD330N(Zn) the electronic density is distributed between Zn2 and Zn3 through Asp51, and almost truncated between Zn2 and Ser102, supporting the idea that Zn2 does not truly activate Ser102 (Figure 2(a)). In Ê is typical APD153HD330N(Mg), the distance of 2.10 A of the activated conformation of Ser102 as generally observed in other structures of alkaline phosphatase when Ser102 interacts with Zn2. In addition, the electronic density is continuous between Ser102, Zn2, Asp51 and Mg3, revealing the strong interaction between Zn2 and Ser102 (Figure 2(b)). Active site of the complexed APD153HD330N(PO4), and APD153HD330N(AlF3) When a crystal is soaked in the presence of phosphate, a large density peak of 17s is observed in the Fo ÿ Fc map, compatible with the presence of a phosphate ion (APD153HD330N(PO4)). This phosphate ion occupies the same position as in AP, APD330N, or APD153H. Ser102 Og interacts with Thr155, and has rotated by 140  around its Ca-Ca bond from the activated conformation observed in the free enzyme (Figure 3(a)). When a crystal is soaked in the presence of NaF and AlCl3, the electronic density map reveals the presence of a 12s peak centred on the metal compatible with the presence of AlF3 (APD153HD330The density indicates a trigonal N(AlF3)). bipyramidal AlF3 with Ser102 Og and a water molecule as coaxial ligands as in the case of APD330g Ê N(AlF3) (Figure 3(b) and (c)). Ser102 O is 1.9 A from the aluminium ion, and has rotated by 30  around its Ca-Ca bond as compared to the free Ê from enzyme. The apical water molecule is 1.8 A the aluminium ion. Furthermore, AlF3 occupies a position that is similar to that observed with the phosphate ion in APD153HD330N(PO4). As compared to APD153HD330N(PO4), the apical water molecule can be assimilated to the fourth oxygen atom of Ê and 1.8 A Ê on the phosphate. The distances of 1.7 A either side of the aluminium ion are compatible with an associative transition state, as in the case of APD330N(AlF3). In both APD153HD330N(PO4) and APD153HD330N(AlF3), the hydrogen bond network included Wat1, Wat2 and Wat4 at the same locations as in APD330N. Wat3 is absent but this is probably due to the absence of a well-de®ned metal ion at M3. An electron density peak is visible in both complex structures but, as shown in Table 4, the peak height is very small. In addition, the orientation of the histidine residue and the environment of the peak are incompatible with an octahedral coordi-

Arti®cial Evolution of E. coli AP Active Site

947

Figure 2. (a) Ball and stick representation in stereo view of Asp51, Ser102, Zn2 and Zn3 in the active site of APD153HD330N(Zn), and 1s contoured 2Fo ÿ Fc map. (b) Ball and stick representation in stereo view of Asp51, Ser102, Zn2 and Mg3 in the active site of APD153HD330N(Mg), and 1s contoured 2Fo ÿ Fc map.

nation. We therefore concluded that these small peaks could correspond to the presence of residual zinc at site M3, or to a water molecule. The magnesium at M3 is therefore highly unstable, probably due to the introduction of His153, as histidine is known for its high af®nity toward zinc. In order to improve the enzyme stability, we removed the residue side-chain at position 153 and substituted histidine by glycine. Active site of APD153GD330N(Mg) and APD153GD330N(PO4) When a crystal of APD153GD330N is soaked in the presence of 10 mM magnesium and 20 mM zinc, the M3 site is occupied by an octahedral magnesium ion, revealing that the af®nity of the magnesium is higher than that of zinc for this site. When a crystal of APD153GD330N is soaked in the presence of phosphate and magnesium, the electronic density map reveals that the M3 site is still occupied by magnesium, and that the phosphate ion is present in the active site (Figure 4). In order to trap the phosphate ion in the active site, the soaking times needed to be very long, as shown in Table 2. As a consequence of this long soaking time, the peak heights in the Fo ÿ Fc map are smaller than in other complexes, re¯ecting the fact that the occupancy of these ions is probably not 100 %.

However, the B-factors of the metal and phosphate ions after re®nement with full occupancy are reasonable (i.e. <50 A2), and the electronic density con®rms the presence of the ions in this structure. The distance between the phosphate ion and the Ê ), and the continuous electronic seryl moiety (1.8 A density correspond to a covalent bond. Therefore, in the presence of phosphate in the soaking solution, we observe the phosphoseryl intermediate of APD153GD330N(PO4) after a long soaking time, and the non-covalent enzyme phosphate complex could never been isolated. In addition to the covalent bond with Ser102, the phosphate ions interact with Arg166, with the two zinc atoms, and with Wat2. Three water molecules out of the four observed in APD330N are present in APD153GD330N (PO4), Wat1 is not observed. Wat2 bridges the phosphate ion to Lys328. Wat3 occupies approximately the position of Asp153 carboxylate in Ê away from the position APD330N, and is located 3 A of the corresponding Wat3. It interacts with Wat4, and Arg166, but not with the phosphate moiety or with the magnesium atom. Wat4 bridges Arg166 to Wat3. The hydrogen bond network is weak, as in the case of APD153HD330N, but the magnesium ion clearly occupies the M3 site. This con®rms that in APD153HD330N the histidine residue at position 153 tends to trap a zinc atom at M3, which probably contributes to lowering the activity of the enzyme.

948

Arti®cial Evolution of E. coli AP Active Site

Figure 3. (a) and (b) Ribbon, and ball and stick representation of the active site of APD153HD330N and of the interactions that involve residues Asp51, Ser102, His153, Thr155, Arg166, Glu322, Lys328, water molecules Wat1 to Wat4, and Zn1 and Zn2. (a) In the presence of phosphate in the active site. (b) In the presence of aluminium ¯uoride in the active site. (c) Ball and stick representation in stereo view of the active site of APD153HD330N with aluminium ¯uoride in the active site, and 1s contoured 2Fo ÿ Fc map.

Discussion Here, we present the structural aspect of a combined approach of mutagenesis and structural analysis that leads to a double point mutant of alkaline phosphatase merging both E. coli and bovine enzyme qualities with regard to the colorimetric properties of the protein.12 Our goal was not to detail the catalytic mechanism of this enzyme, which is already well known, but to perform a step by step structural analysis of different mutants in order to point out the structural events associated with the evolution of the enzyme properties. The ®rst step, based on random mutagenesis, let us isolate a single point mutant, D330N.3 The active-site structure of this mutant is similar to that of AP, and Asn330 superimposes perfectly on Asp330 of AP. The activity enhancement observed for this mutant should therefore be attributed to a long-distance effect that may include the shift of local pH induced by the substitution. Four water

molecules seem to play an important role in the phosphate stabilisation in the non-covalent enzyme-phosphate complex as well as in the preceding transition state mimicked here with AlF3 (Figure 1). Wat1 is positioned in the same region as the nucleophilic water molecule described previously.18 Wat2 bridges the phosphate ion (or AlF3) to Lys328. The Mg-bound water molecule Wat3 might play the role of a general base in the generation of the Ser102 nucleophile, and of a general acid in the regeneration of the Ser102 hydroxyl group as described.14 Wat4 interacts with Arg166, and therefore reinforces the hydrogen-bond network PO4-Arg166-Asp153-Lys328-Wat2-PO4. The similarity of the hydrogen bond network of the active site between the two complexes APD330N (PO4) and APD330N(AlF3), including the water molecule positions, suggests that the transition from the phosphoseryl state to the non covalent enzyme-phosphate complex might be very easy, since no conformational adjustment is needed.

Arti®cial Evolution of E. coli AP Active Site

949

Figure 4. (a) Ribbon, and ball and stick representation of the active site of APD153GD330N and of the interactions that involve residues Asp51, Ser102, Gly153, Thr155, Arg166, Glu322, Lys328, water molecules Wat1 to Wat4, and Zn1, Zn2 and Mg3, in the presence of phosphate in the active site. (b) Ball and stick representation in stereo view of the active site of the phosphoseryl intermediate of APD153GD330N, and 1s contoured 2Fo ÿ Fc map.

However, the high level of stability observed in the non-covalent enzyme-phosphate complex explains the limiting character of the phosphate release step. In addition, the active site of these complexes superimposes fully on that of the wild-type enzyme. In particular, the transition state is probably similar in the wild-type and in APD330N, as the structure of wild-type AP in complex with the transition state analogue vanadate superimposes perfectly on that of APD330N (AlF3), and the three water molecules Wat1, Wat2 and Wat3 are present at the same location in both structures. This high level of preservation between the wild-type enzyme and this ®rst mutant prevents us from understanding the reason for the activity enhancement of the enzyme. However, mutation D330N introduces an important pKa modi®cation located next to His331, in interaction with Zn2, and close to Lys328, involved in the phosphate stabilisation. The crucial point for our purpose is that the mutation D330N does not affect the structure of the enzyme, and APD330N is, therefore, a structural platform as good as the native enzyme for additional mutations, but with a better catalytic activity. At this point, the approach to further increase catalytic activity was to mutate a residue involved in the phosphate stabilisation. We targeted position 153 because it is part of the hydrogen bond network stabilising the phosphate ion, it interacts with two water molecules among the four

described above, it is not conserved in the intestinal bovine enzyme, and it leads to an enzyme that is already threefold more active than the wild-type when activated by magnesium.24 The substitution D153H together with the initial substitution D330N lead to our ®rst highly active mutant with a kcat 17fold higher than that of the wild-type enzyme. Interestingly, APD153HD330N is activated by the magnesium ion like the APD153H mutant. On the other hand, APD153HD330N is unstable, and if the initial activity is very high, an important drop is observed after few days.3 In order to understand the properties of this mutant, we analysed its structure in four different states. In the absence of phosphate, we analysed the structure in the presence of magnesium or zinc in the soaking solution. These structures reveal that the magnesium activation process of the catalytic Ser102 proceeds through a continuous electron density from Mg3 to Ser102 via Asp51 and Zn2 (Figure 2). In the presence of zinc at M3, this electron continuum remains only between Zn2 and Zn3, and is broken between Zn2 and Ser102. This observation con®rms the importance of the presence of magnesium at M3 in order to have a fully active enzyme with Ser102 being activated by Zn2. In the presence of phosphate or AlF3, the weakness of the electronic density at the M3 site does not permit modelling of any atom. Since the magnesium ion is crucial for the activation of the catalytic serine residue, the instability of the metal ion at M3 could explain the drop of

950 the enzyme activity observed after few days. The location of site M3 close to position 153 suggests that the mutation D153H might be partially responsible for the lack of stability of this mutant. The high af®nity of histidine for zinc may favour the presence of this inhibitor metal at M3. Moreover, in both APD153HD330N(PO4) and APD153HD330N(AlF3), there is no direct interaction of phosphate or AlF3 with K328, as was the case in the single point mutant D153H.24 The phosphate coordination includes Wat2. Finally, the stabilising hydrogen bond network is weaker than in APD330N or in the wild-type enzyme, allowing the phosphate ion to leave the active site more easily. We introduced the substitution D153G instead of the substitution D153H together with the substitution D330N, which lead to a 40-fold more active mutant in the presence of the phosphate acceptor diethanolamine, as compared to the wild-type enzyme. The structure of this mutant contains a magnesium ion at site M3 after both soaking conditions. APD153GD330N is activated by magnesium like APD153HD330N, but the af®nity of magnesium for M3 seems to be partly restored. It is therefore tempting to postulate that the stability of APD153GD330N activity may be correlated to a higher af®nity of M3 for magnesium than for zinc and, above all, that the lack of stability of APD153HD330N activity was the consequence of higher af®nity of M3 for zinc than for magnesium. The major point concerning APD153GD330N is the observation of the intermediate phosphoseryl state when phosphate is added to the soaking solution. This intermediate state has not been observed with the wild-type enzyme, D330N, D153H or D153G mutants or with any mutant harboring an improved catalytic activity.8 It has been reported only for APWT inhibited with cadmium,6 and for the weakly active mutant H331Q.18 In both cases, the phosphoseryl intermediate was observed because the phosphoenzyme intermediate was stabilized. On the contrary, in our case, the phosphoseryl intermediate is observed in the most active mutant ever reported for E. coli alkaline phosphatase. This possibly re¯ects the accumulation of the covalent intermediate at equilibrium instead of the non-covalent enzyme-phosphate complex. This may re¯ect a change in the relative stability of the covalent enzyme-phosphate complex versus the noncovalent complex. This would mean that the noncovalent complex that corresponds to the more stable state in APWT would be less stable than the covalent complex in APD153GD330N. It is not possible to dissociate the effect of each mutation individually, but it is interesting to notice that in the single point mutant D153H the breakdown of the hydrogen network R166-D153-K328 is compensated partially by a direct interaction between K328 and the phosphate ion.24 In all our mutants, Wat2 is always present and bridges K328 to the phosphate ion, as in the wild-type enzyme. We may therefore envisage that one of the effects of the mutation D330N would prevent K328 from

Arti®cial Evolution of E. coli AP Active Site

switching toward the phosphate ion and forming a direct interaction. In conclusion, the combined approach of mutagenesis and structural analysis allowed us to obtain an alkaline phosphatase that includes advantages of both the bacterial and the mammalian enzyme with regard to colorimetric tests. Thus, it is easy to express alone or fused,31 it is very stable as E. coli AP, and it displays an activity level similar to that of the most active alkaline phosphatase; namely, bovine intestinal AP. Moreover, the evolution of kcat and Km of the E. coli enzyme to levels similar to those of the bovine enzyme raises the question of whether it is a coincidence or a necessary consequence.

Materials and Methods Expression and purification of alkaline phosphatases Mutant enzymes were expressed as described.3 Brie¯y, transformed cells were grown overnight at 37  C in LB medium supplemented with 200 mg/ml of ampicillin and then seeded to a 1/50 dilution in 200 ml of lowphosphate medium.32 After ®ve hours of incubation at 37  C, the periplasmic proteins were extracted from the bacterial culture by cold osmotic shock as described.33 The resulting periplasmic extracts were concentrated from 20 ml to 1 ml and dialysed against 0.02 M Tris-HCl (pH 8.0), 1 mM MgCl2 at 4  C by use of a Centricon-30 microconcentrator. Chromatofocusing of the APs was performed on a fast protein liquid chromatograph (FPLC) system by use of a Mono P HR 5/5 column and Polybuffer 74 and 96 as described by Mandecki et al.8 Active fractions were analysed by SDS-PAGE.34 Fractions containing a single 48,000 Da band were pooled and concentrated to 100 ml. Polybuffers were removed on Sephadex G-75 equilibrated in 20 mM Tris-HCl (pH 8.0), 1 mM MgCl2. Electrophoresis on SDS-PAGE revealed a single major band that represented more than 95 % of the total stained protein. The enzymes were stored at ÿ20  C in 20 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.02 % (w/v) NaN3, 50 % (v/v) glycerol. Concentrations of the puri®ed wild-type and mutant enzymes were determined by absorbance measurements at 278 nm with an extinction coef®cient of 0.71 cm2/mg.35 Assays of enzyme activity Enzyme activity measurements were performed at 25  C in the presence of Caps buffer, or at 37  C in the presence of diethanolamine. The enzymatic activity was measured spectrophotometrically using p-nitrophenyl phosphate (pNPP) as substrate35 by monitoring the release of p-nitrophenolate (pNP) at 410 nm (molar extinction coef®cient of pNP 1.62  104 Mÿ1 cmÿ1). Buffers used were 0.1 M Caps (pH 10.0), 10 mM MgCl2, 0.4 M NaCl or 1 M diethanolamine (pH 10), 10 mM MgCl2, 20 mM ZnCl2, 0.5 M NaCl. Initial rates were calculated and values of Vmax and Km were obtained by ®tting the data to a linear function (Eadie & Hofstee plot). The kcat values were computed from the Vmax values using a dimer molecular mass of 96,000 Da. To measure the magnesium activation, the enzyme was diluted to 480 ng/ml and preincubated in a solution of 50 mM Tris-HCl (pH 8.5), 10ÿ5-10ÿ1 M MgCl2 for two hours at 25  C. The enzymatic activity was measured spectrophotometrically

951

Arti®cial Evolution of E. coli AP Active Site using pNPP as substrate.36 The release of pNP at 410 nm (molar extinction coef®cient of pNP 1.62  104 Mÿ1 cmÿ1) was monitored after ten minutes of substrate incubation at 25  C in the presence of Caps buffer, or at 37  C in the presence of diethanolamine. Crystallisation The sitting drop method was used to grow crystals by vapour diffusion. The crystallisation condition reported previously6,26 had to be adjusted for the AP mutants. The optimal conditions included 2.4 M (NH4)2 SO4, 10 mM MgCl2, 1 mM ZnSO4, 100 mM Tris-HCl (pH 8.0), with a protein concentration of 30-40 mg/ml. This corresponds to a zinc concentration 100 time higher than in the conditions used for the wild-type enzyme, and a pH 1.5 unit lower. In these conditions, crystals of 0.3 mm  0.3 mm  0.3 mm in size were grown in six to eight weeks at room temperature, in a 30 ml drop. Crystal soaking A ®rst crystal of APD330N was mounted directly from its drop without any soaking (APD330N(PO4)). A second crystal of APD330N was soaked for three days in 2.2 M (NH4)2SO4, 0.05 M Tris-HCl (pH 8.0), 10 mM MgCl2, 25 mM NaF, 0.5 mM AlCl3 (APD330N(AlF3)), as described.37,38 One crystal of APD153HD330N was soaked in 2.4 M (NH4)2SO4, 0.05 M Tris-HCl (pH 8.0), 10 mM MgCl2, 0.1 mM ZnSO4 (APD153HD330N(Zn)). To restore the magnesium at M3, the zinc salt was removed from the soaking condition, and the magnesium concentration was brought to 100 mM. Therefore, the initial soaking contained 2.4 M (NH4)2SO4, 0.05 M Tris-HCl (pH 8.0), 100 mM MgCl2 (APD153HD330N(Mg)). To observe the phosphate ion in the active site, a second soaking solution was prepared with 2.4 M (NH4)2SO4, 0.05 M Tris-HCl (pH 8.0), 10 mM MgCl2, 25 mM NaH2PO4 (APD153HD330N(PO4)). Finally, to observe the aluminium ¯uoride molecule in the active site, a third soaking solution was prepared with 2.4 M (NH4)2SO4, 0.05 M TrisHCl (pH 8.0), 10 mM MgCl2, 25 mM NaF, 0.5 mM AlCl3 (APD153HD330N(AlF3)). The magnesium concentration had to be lowered to 10 mM in solutions containing phosphate ion or aluminium ion to avoid the formation of a precipitate. One crystal of APD153GD330N was soaked in 1.1 M (NH4)2SO4, 0.05 M Tris-HCl (pH 8.0), 10 mM MgCl2, 20 mM ZnSO4 for one day (APD153GD330N(Mg)). A second crystal was soaked sequentially in a solution containing 2.2 M (NH4)2SO4, 0.05 M Tris-HCl (pH 8.0), 50 mM MgCl2 for six months, and then in a solution containing 2.4 M (NH4)2SO4, 0.05 M Tris-HCl (pH 8.0), 10 mM MgCl2, 100 mM NaH2PO4 for six months (APD153GD330N(AlF3)). All the soaking condition are summarised in Table 2. Data collection and processing Diffraction data corresponding to APD330N(PO4), APD153HD330N(Zn), and APD153HD330N(Mg), were collected Ê , 2.6 A Ê , and 2.5 A Ê resolution, respectively, on a to 2.4 A CCD detector on station D2AM at the ESRF. The data were processed and integrated with the program XDS.39 Diffraction data corresponding to APD330N(AlF3), APD153HD330N(PO4), APD153HD330N(AlF3), and Ê , 2.5 A Ê , 2.3 A Ê, APD153GD330N(Mg), were collected to 2.0 A

Ê resolution, respectively, on a 300 mm diameter and 2.4 A Marresearch imaging plate system mounted on a Rigaku H2R rotating anode. The data were processed with Denzo and integrated with Scalepack from the HKL Diffraction data corresponding to package.40 Ê resolution on APD153GD330N(PO4), were collected to 2.5 A a 300 mm diameter Marresearch imaging plate system at LURE station DWat32. The hexagonal space group P6322 was determined by using the autoindexation program included in XDS, and by visual inspection of the extinction on pseudo-precession images built with the program Hklview from CCP4.41 The statistics of the data collection and processing are summarised in Table 3. Structure solution The structure was solved by molecular replacement with AMoRe,28 using the data of APD330N(PO4) from the Ê resolution. As the trial model, ESRF, truncated to 3.0 A the coordinates of the wild-type E. coli AP (PDB entry pdbalk1.ent) were used, including metal and phosphate ions. The ®rst solution after rotation had a peak height at 2.5s above any other peak, giving a solution corresponding to one monomer in the asymmetric unit. The translation search con®rmed the enantiomorphic space group P6322. After translation, the correlation of the ®rst solution was 68.9 and the R-factor was 33.3 %. After the ®rst rigid-body re®nement, the correlation was 72.3 and the R-factor was 31.7 % (Table 3). Structure refinement In the ®rst stage of the re®nement, in each monomer the metal ions, phosphate ions and residues 1 to 15 were removed, and residues 51, 101-103, 153, 155, 166, 322, 327 to 331, 369, 372 and 412 from the active site were substituted by alanine. All crystal structures were re®ned using XPLOR version 3.1.29 Each cycle of standard re®nement included positional re®nement, simulated annealing, and temperature-factor re®nement. In the initial stages of the re®nement, non-crystallographic symmetry constraints were introduced. After each cycle of re®nement, the model was visualised and modi®ed with the help of the program TURBO-FRODO.42 Water molecules were added by visual inspection of the Fo ÿ Fc map for peaks higher than 3.5s with a reasonable distance from the protein. After re®nement, the geometry was checked with PROCHECK.43 The statistics of the re®nement are summarised in Table 2. The four models were superimposed on the wild-type model using the main-chain atoms with the iterative program ALIGN.44 Protein Data Bank accession codes The coordinates have been deposited in the RCSB Protein Data Bank: the ID codes are 1KH5, 1KH4, 1KHN, 1KHK, 1KHL, 1KHJ, 1KH7 and 1KH9 for APD330N(PO4), APD330N(AlF3), APD153HD330N(ZN), APD153HD330N(MG), APD153HD330N(PO4), APD153HD330N(AlF3), APD153HD330N (MG) and APD153HD330N(PO4)), respectively.

Acknowledgments We thank Dr M. Roth and Dr J. L. Ferrer for their help in the data collection at the ESRF. # 2002 CEA

952

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Edited by R. Huber (Received 10 September 2001; received in revised form 18 December 2001; accepted 21 December 2001)