Structural and Enzymatic Properties of 1-Aminocyclopropane-1-carboxylate Deaminase Homologue from Pyrococcus horikoshii

doi:10.1016/j.jmb.2004.06.062

J. Mol. Biol. (2004) 341, 999–1013

Structural and Enzymatic Properties of 1-Aminocyclopropane-1-carboxylate Deaminase Homologue from Pyrococcus horikoshii Aiko Fujino1, Toyoyuki Ose1, Min Yao1, Tetsuo Tokiwano2 Mamoru Honma3, Nobuhisa Watanabe1 and Isao Tanaka1* 1

Division of Biological Sciences Graduate School of Science Hokkaido University, Sapporo 060-0810, Japan 2 Division of Chemistry Graduate School of Science Hokkaido University, Sapporo 060-0810, Japan 3

Division of Applied Bioscience Graduate School of Agriculture Hokkaido University, Sapporo 060-8589, Japan

1-Aminocyclopropane-1-carboxylate deaminase (ACCD) is a pyridoxal 5 0 -phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield a-ketobutyrate and ammonia. We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms. In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3. However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity. Instead of ACCD activity, the GC–MS analysis and enzymatic method show that phAHP has deaminase activity toward L and D-serine. Here, we present the crystal structures of the native and ACC-complexed phAHP. The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site. Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins. We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5 0 -phosphate (PLP), not the difference of the catalytic residues themselves. We also propose a condition necessary in the primary sequence to have ACCD activity. q 2004 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: 1-aminocyclopropane-1-carboxylate; pyridoxal 5 0 -phosphate; genome annotation; crystal structure

Introduction 1-Aminocyclopropane-1-carboxylate deaminases (ACCDs) isolated from a few strains of Pseudomonas species,1–4 the rhizobacterium Enterobacter cloacae,5 Abbreviations used: PLP, pyridoxal 5 0 -phosphate; ACC, 1-aminocyclopropane-1-carboxylate; ACCD, 1aminocyclopropane-1-carboxylate deaminase; phAHP, ACC homologue from Pyrococcus horikoshii; hACCD, 1aminocyclopropane-1-carboxylate deaminase from the yeast Hansenula saturnus; TRPSb, b-subunit of tryptophan synthase; PH0054, the protein expressed in Escherichia coli from open reading frame (ORF) ph0054 of the hyperthermophilic archaea Pyrococcus horikoshii OT3; AHP, ACCD homologue protein; GC–MS, gas chromatography–mass spectroscopy; r.m.s.d., root-meansquare deviation.

the yeast Hansenula saturnus6 and the fungus Penicillium citrinum7 have the ability to degrade ACC into a-ketobutyrate and ammonia (Figure 1). We have already reported the crystal structures of ACCD from the yeast H. saturnus (hACCD) in the absence and presence of ACC.8,9 The reaction mechanism for opening the cyclopropane ring of ACC was discussed from the complex structures. The essential residues for catalysis and ACCrecognition were derived from their structures and site-specific mutational analysis. The amino acid sequence alignment of several enzymes defined as ACCD (including putative ones) is shown in Figure 2. This alignment shows that most of the important residues are conserved. For example, the lysine residue (Lys51 in hACCD) that is bound to pyridoxal 5 0 -phosphate (PLP) with the

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

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Structural and Enzymatic Properties of ACCD

Figure 1. Reaction scheme of ACCD.

Schiff base (ACCD requires PLP or vitamin B6 as a cofactor for its catalysis), the tyrosine residue (Tyr295) that stacks to the pyridine ring of PLP and promotes to form the “external” Schiff base of PLP-ACC, and the serine residue (Ser79) to recognize the ACC are important for ACCD reaction.9 The PLP enzymes are the most versatile biocatalysts, involved in a wide range of metabolic reactions. Our previous structures for several states of hACCD have suggested a novel reaction mechanism for ACC degradation. However, little is known about the physiological roles of ACC in bacteria. The compound ACC is a precursor of the plant hormone ethylene, which controls various stages of plant growth and development.10 Initially, ACCD

was discovered from soil bacterium Pseudomonas sp. strain ACP when ACC was the sole nitrogen source for the growth of this bacterium.1,11 Therefore, the possibility of a mutualistic relationship between higher plants and soil bacteria has also been suggested.12,13 Only the fungus P. citrinum has both ACC oxidase and ACCD. ACC is biosynthesized and degraded in this organism.7 In this organism, it was also described that the ACC accumulated in the cells induces the expression of ACCD, thus self-regulating the level of ACC. In addition to these ACCDs, several open reading frames (ORFs) in fully decoded organisms are annotated as ACCD homologues with significant sequence identity. One of them, the protein PH0054 from the hyperthermophilic archaebacterium

Figure 2. Amino acid sequence alignment of the annotated ACCDs including those of genuine ACCDs. The sequences displayed are from P. horikoshii (PH0054: phAHP), P. abyssi, E. coli, Pseudomonas sp. ACP (pACCD), H. saturuns (hACCD), and E. cloacae (eACCD). All sequences were obtained from the SWISS-PROT protein sequence database. The sequential numbers above and below the alignment represent the numbers of phAHP and hACCD. The residues highlighted in red represent complete conservation; those in blue represent conservative mutations; and those in green represent allowed mutations. The lysine residue that probably binds to PLP with the Schiff base is marked with a yellow circle. The residues that seem to approach the pyridine nitrogen atom of PLP are marked with purple circles. The identity scores of each sequence versus PH0054 are also shown here.

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Structural and Enzymatic Properties of ACCD

Pyrococcus horikoshii OT3,14 was predicted to be ACCD. The sequence of PH0054, in addition to other annotated homologues, preserves the key amino acid residues (Figure 2). However, hyperthermophilic archaebacteria such as P. horikoshii live under extreme circumstances, where higher plants cannot survive. Therefore, the function of this protein in archaebacteria remains to be elucidated. In the present analysis we have over-expressed the ACC homologue from P. horikoshii (phAHP) in Escherichia coli, characterized its enzymatic activity, and determined the crystal structure in the presence/absence of ACC. The thermophilic enzymes have been studied extensively, and it has been reported that the optimal temperatures for their activity are around those under which they live. Therefore, phAHP had been expected to show deaminase activity toward ACC at about 98 8C, which is the optimum growth temperature for P. horikoshii. However, phAHP did not show ACC deaminase activity at high temperature nor at room temperature. Information on the three-dimensional structure of the enzyme is indispensable to an explanation of the inertness of phAHP for ACC. The crystal structures of native and ACC complexed phAHP, which can clarify the features behind this inertness, are also supported by several structures of hACCD variants and mutagenesis analysis.

Results and Discussion The enzymatic activity of phAHP Despite the sequence similarity between ACCD from yeast H. saturnus or bacteria belonging to some Pseudomonas species, phAHP did not show any deaminase activity toward ACC at high (80 8C) or room (30 8C) temperatures. Rather, it showed deaminase activity toward D and L-serine, and produced private. The specific activity at 80 8C for D and L -serine is 2.88 (units/mg) and 0.54 (units/mg), and at 30 8C is 0.74 (units/mg) and 0, respectively. The comparison of enzymatic activity between authentic ACCD (Pseudomonas sp. ACP1,11) and phAHP is summarized in Table 1. The deaminase activity for each substrate shows clear dependency on reaction temperature; the activity for L-serine at 80 8C is five times larger than at 30 8C. The absorption spectra of phAHP with and without ACC were measured in 50 mM potassium phosphate (pH 7.5). The absorption Table 1. The specific activity of authentic ACCD (Pseudomonas sp. ACP) and phAHP

ACCD, 30 8C phAHP, 30 8C phAHP, 80 8C

ACC (units/mg)

L-Serine (units/mg)

D-Serine (units/mg)

5.24 0 0

0 0 0.54

0.1 0.74 2.88

spectrum of native AHP gives a maximum at 420 nm, typical of PLP enzymes, due to the internal aldimine of Lys54-PLP. In the presence of ACC, the absorption maximum was shifted to 435 nm, suggesting that an external aldimine of ACC-PLP was formed. (For the reaction, 3.3!10K2 mM phAHP and 0.1 M ACC at a ratio of 1 : 1 were mixed and incubated at 80 8C for ten minutes.) Protomer structure According to the assignment of secondary structure by DSSP,15 the protomer of phAHP includes 13 a-helices, ten b-strands, and two 310 helices. The phAHP protomer (Figure 3(a)) consists of a large and a small domains. The small domain (residues 54–160) is composed of four a-helices and four parallel b-strands. The core of the domain is folded as a twisted a/b sheet structure. The N-terminal helix (helix 1) exists apart from the core region. The large domain (residues 1–53 and 161–325) is composed of nine a-helices and six b-strands, which are folded as a twisted a/b sheet structure resembling the small domain. All strands excepting the second one (bB) in the b-sheet of the large domain run parallel. The bstrand core is surrounded by five helices (from a7 to a11). The N-terminal a1 helix and the C-terminal a12 and a13 helices exist distantly from the twisted a/b core separated by loops. There are two series of short a-helices at the C terminus, located at the opposite side of the N terminus in the large domain. Four parallel b-strands (bC-bF) in the small domain are sandwiched between one helix (a6) and three helices (a3-a5). The coenzyme PLP is positioned in the interfacing crevice between the small and large domain with the Schiff base to Lys54, at the tip of the helix a3. The crevice is surrounded by polar residues. The folding topology of phAHP is basically similar to that of hACCD; the Ca atoms can be ˚) superimposed well (r.m.s. deviation is 2.0 A (Figure 3(b)). The identity of amino acid sequence between phAHP and hACCD is about 30%, although the two proteins show different enzymatic activities. Secondary structural elements of these proteins are given in Figure 4 with sequence alignment. The most prominent difference observed in the whole structure of phAHP as compared with that of hACCD is the lack of the loop A region (104–116) and loop B region (168–171) in the former (Figure 3(b)). There are no corresponding residues in the sequence of phAHP. In the structure of hACCD, hydrogen bonds between Glu109 on this loop A with Ser333 and Ser334 (nearly C-terminal region) are observed. These interactions, which cannot be formed in phAHP because of the lack of the above loop regions, play a role in stabilizing both domains in hACCD. A cavity engraved profoundly from the molecular surface to the active site is also formed around this loop region. The cavity located in hACCD is not identified in the structure of phAHP,

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Structural and Enzymatic Properties of ACCD

Figure 3. Protomer structure of phAHP. (a) Ribbon diagram displayed in different colors: large domain (blue), small domain (orange), and PLP (yellow) that locates at the inter-domain crevice. The a-helices are numbered a1, a2,. and bstrands are designated bA, bB,. from the N terminus. (b) Least-squares Ca trace superposition of phAHP (blue) and hACCD (green). Loop A and loop B of hACCD are not present in the structure of phAHP. PLP is omitted from the hACCD structure (stereo view).

which renders the substrate easily accessible to the active site. Dimeric structure and crystal packing Three independent protomers of phAHP molecules (named A, B, and C) are packed in the asymmetric unit of the native-type P3221 crystal (Figure 5). All protomers form dimers with 2-fold rotation relationships. One of the dimers (AA dimer) is formed with the crystallographic 2-fold relation, whereas the B and C protomers form a dimer (BC dimer) with a non-crystallographic 2fold. In the interface of the BC dimer, 12 water

molecules are assigned. The inter-protomer interactions are hydrogen bonds between these 12 water molecules and 13 residues. On the other hand, the AA dimer has 20 inter-protomer hydrogen bonds involving 11 water molecules. Six of them are at the same positions as those found in the BC dimer. In addition, near Arg17 of the A protomer, one 2propanol molecule was assigned from the electron density map. The 2-propanol molecule was contained in the crystallization reagents. The AA dimer and the BC dimer can be superimposed well, except for the slight positional change of the Trp20 indole side-chain. The r.m.s. deviation value between AA ˚ for main chains and 0.732 A ˚ and BC is 0.487 A

Structural and Enzymatic Properties of ACCD

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Figure 4. Amino acid sequence alignment of phAHP, hACCD, and ACCD from Pseudomonas sp. ACP (pACCD) with secondary structures. The sequential numbers and secondary structures above the alignment represent the numbers of PH0054, below is hACCD. The residues highlighted in red represent complete conservation; those in green represent conservative mutations; and those in blue represent allowed mutations. The secondary structures of phAHP and hACCD are separated into the large (blue) and the small (orange) domain. The secondary structure parts are named a1a13 and bA-bJ, and 310 helices are I and II. The dotted lines represent the deletion region in each sequence. The deleted loop regions in phAHP that exist in hACCD are shown in purple (loop A and loop B).

for side-chains. The assembly of the phAHP dimer is similar to that of the hACCD dimer. Structure around PLP of native AHP The milieu around the active site is shown in Figure 6. As is the case for the other PLP enzymes, the active site exists in the bottom of the crevice between two domains (Figure 3). In the active site of native phAHP, the PLP is bound to Lys54 with the Schiff base between C4 of PLP and the side-chain amino nitrogen atom of Lys54. One sulfate ion added in the crystallization condition was observed in the active site. The sulfate ion was also observed in the native structure of hACCD.8 The pyridine ring of PLP is stably stacked by the aromatic ring of Tyr282, and the phosphate group of PLP is encircled by Lys57 and glycine-rich region (residues Ser191, Gly192, Gly193 and Thr194) from the large domain and thus determined its direction. The pyridine ring nitrogen atom of PLP exists within the distance of ˚ ) with the side-chain hydrogen bonding (2.6 A hydroxyl oxygen atom of Thr308. The residue arrangements around PLP in the active site are similar between phAHP and hACCD.

Substitutions of residues around the active site are only His80(phAHP)-Gln77(hACCD), Thr283 (phAHP)-Glu296(hACCD), and Thr308(phAHP)Leu323(hACCD) (Figure 6). The side-chain directions of His80(phAHP) and Gln77(hACCD) are completely different. His80(phAHP) is a key residue for domain closure in the complexed form as will be described later, and the difference may be critical for protein function. Thr308(phAHP) is located within distance of the hydrogen bond to the pyridine nitrogen atom of PLP. The corresponding residue of hACCD (Leu323) has completely different characteristics and directs differently (Figure 6(b)). Alternatively, in the hACCD structure, the carboxylate oxygen atom of Glu296 makes a hydrogen bond ˚ .8 with pyridine nitrogen atom at a distance of 2.5 A The existence of the carboxylate group in this position is widely seen in PLP-dependent enzymes, except the other members of the TRPSb family or alanine racemase from Bacillus stearothermophilus.16 In the TRPSb family, a hydroxyl group such as a serine or threonine side-chain is always found within hydrogen-bonding distance of the pyridine nitrogen atom, as is the case in phAHP. The corresponding residue in phAHP is Thr283, whose

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Figure 5. Ribbon representation of the two dimers. The crystallographic dimer (AA dimer) is shown in red, the non-crystallographic (NCS) dimer (BC dimer) in blue and green. The PLP is shown in yellow using the ball-andstick model. The Figure is a view from the NCS 2-fold axis.

side-chain hydroxyl group faces inversely and cannot interact with the pyridine nitrogen atom. In other words, Thr308 interacts with the pyridine nitrogen atom in the space that the shorter residue (Thr283) provides. In the hACCD structure, such a space is not available because of the long side-chain of Glu296. Interestingly, we have already reported that the E296Q mutation of hACCD loses ACC deaminase activity.9 Conformational change upon ACC binding The crystal structure of phAHP complexed with ACC was also analyzed in this study. The space group of the complex crystal was P1 with 24 protomers per asymmetric unit. Figure 7 shows the superposition of Ca traces of native and ACCcomplex structures. A swinging movement between inter-domains was observed upon ACC binding. The movement was evaluated in the following way. (1) Least-squares superposition of

Structural and Enzymatic Properties of ACCD

all the Ca atoms (residues 1–325) between structures of native protomer A and complexed protomer A was executed using program LSQKAB.17 (2) The residues whose r.m.s. deviation values are higher than 0.63 were removed, and the operator was recalculated to superpose the structures. (The r.m.s. deviation values of the three monomers in the native crystal were 0.63 on average; therefore, 0.63 was used as the threshold in order to exclude the residues that ought not to be used.) This process was repeated until all the r.m.s. deviations of the respective residues used in the current calculation became lower than the threshold. (3) The residues surviving this sieve process were considered not to have moved significantly, and they were used for calculating a conversion matrix for superposing the two structures (Figure 7). The r.m.s. deviation between native phAHP and ACC-bound structures indicates a large movement in the region of residues 64–153. All of these residues are located in the small domain and are grouped into three parts (regions I–III; Figure 8). This r.m.s. deviation curve shows a trend almost similar to that of the B-factor versus residue number plot drawn in Figure 8. In particular, the B-factors of the atoms involved in region III are very high ˚ 2). The B-factor distribution of (sometimes over 80 A both main chains is indicated by rainbow-color gradation in Figure 7. The correlation of r.m.s. deviation and the B-factor can be seen in this Figure. In fact, the sA-weighted FoKFc map around this region is somewhat obscure. Therefore, this region is expected to allow freedom of movement. The region consisting of residues 217–223 of the large domain also shows a very large r.m.s. deviation in Figure 8. This region constitutes a part of helix a9 and is exposed to the crevice between the two domains, which exists at the entrance toward the active site (Figure 3). The conformational change of phAHP, as we call domain closure upon ACC binding, covers the active site. The phenomenon of domain closure is observed in many enzymes, based on analysis of native and substrate complex structures. Structural comparison around PLP The active site of phAHP complexed with ACC is shown in Figure 9. The sA-weighted FoKFc map (contoured at 2.1s) calculated using refined phAHP clearly shows the ACC moiety bound with PLP. In this structure, the side-chain of Lys54 is far from PLP, and the Schiff base between the amino group of ACC and that of PLP (external aldimine) is formed. ˚ The free amino group of the Lys54 is located 4.4 A from the pro-R carbon atom of the ACC. The PLP rotates about 158 between native and ACC complex structures with a spindle from the pyridine nitrogen atom to a phosphate group anchor. The phenol ring of Tyr282 blocks the PLP-pyridine ring to prevent over-rotation; these two planes are nearly parallel. New hydrogen bonds are formed among His80Tyr256-Tyr282 by domain closure (Figure 9). His80

Structural and Enzymatic Properties of ACCD

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Figure 6. The stereo views showing the active site from two different angles ((a) and (b)), after superposition between phAHP (yellow) and hACCD (green). In both structures, PLP molecules form the Schiff base to corresponding lysine residues. The sulfate ion recognized in phAHP is also shown. The residue names and numbers are ordered as phAHPhACCD. The atoms except carbon are colored as follows: oxygen, red; nitrogen, blue; phosphorus, orange; and sulfate, light green.

belongs to the small domain, and Tyr256 and Tyr282 to the large domain. His80 is the only residue that participates in inter-domain bondings, and is important for domain closure.

The superimposition of phAHP-ACC and hACCD(K51T)-ACC complex structures around the active sites is shown in Figure 10(a). Compared to the hACCD(K51T)-ACC structure, few

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Structural and Enzymatic Properties of ACCD

Figure 7. Stereo view showing the comparison between native and ACC complexed phAHP. The translational operator is acquired as described in the text. The ACC complexed structure is indicated by arrows in the Figure. The Ca trace modes are colored according to the B-factor by a rainbow color ramp from red at the highest B-factor values to blue at the lowest B-factor value regions. The regions I, II, and III indicate the portion with higher B-factor explained in the text and for Figure 8.

differences are visible around the active site. The His80 residue described above is a characteristic residue of phAHP; the corresponding residue in the active ACCDs is glutamate (Glu77 in hACCD) (Figure 2). As described, the active site of phAHP is exposed to solvent, and no cavity is present, as observed in the structure of hACCD. Figure 10(b) and (c) present the

accessibility to the active site of these two enzymes. The extra loop region of hACCD (Figure 10(c)) provides aromatic and hydrophobic side-chains such as Trp102 and Leu103, which make the wall of the hydrophobic cavity with other hydrophobic bulky residues such as Tyr113, Phe136, Tyr269, Tyr332 and Phe335, from the molecular surface to the active site.

Figure 8. r.m.s.d. and B-factor plot versus amino-acid residue numbers. A continuous line represents the r.m.s.d. score of the main-chain between native and complexed phAHP and a dotted line represents the main-chain B-factor value of the native structure.

Structural and Enzymatic Properties of ACCD

Figure 9. Active site of ACC complexed phAHP. (a) PLP and ACC are omitted and the map was calculated ˚ , contoured at 2.1s. The C4 0 atom of the PLP and at 2.7 A amino nitrogen atom of the ACC form a covalent bond as an external aldimine. The protein structures with atoms colored for carbon (yellow), nitrogen (blue), and oxygen (red) are shown in gray, the PLP is yellow, and ACC is magenta. (b) The ACC molecule superimposed in the active site of native structure. The pyramid-shaped electron density map (omitted sulfate ion) was calculated ˚ , contoured at 2.3s. (c) Superimposed view of the at 2.5 A native (yellow) and ACC complex (blue) structure.

Insight into inertness The genome-decoding project has uncovered many putative ORFs in the model species. To date,

1007 about 30 ORFs have been annotated as ACCD. However, in these ORFs, only a few proteins have ACC deaminase activity including those from several Pseudomonas species, the rhizobacterium E. cloacae, the yeast H. saturnus and the fungus P. citrinum. The sequence alignment of these ORFs shows at least 20% identity with genuine ACCD (e.g. hACCD), and the lysine residues that are expected to bond to PLP with the Schiff base are fully conserved. Because both hACCD and phAHP have been analyzed in complex with ACC, we are now at the stage of discussing the structure– function relationship in combination with the enzymatic reaction data. The catalytic inertness of phAHP toward ACC is significant because this implies that many putative ACCDs do not have ACCD activity either. We can speculate why this might be, based on a comparison of the two structures. Although phAHP does not have the ability to degrade ACC, it has been found that it reacts to L and D-serine to produce pyruvate. Inertness in response to ACC enabled us to study the reaction mechanism of ACCD in the phAHP structure complexed with ACC without any mutations. The complex structure indicated the existence of ACCPLP external aldimine. This means that phAHP cannot, as proposed for the hACCD structure,9 abstract a b-proton from the pro-R/S carbon atom of ACC. Most of the PLP enzymes retain the basic chemical processes of the PLP catalysis: formation of an external aldimine group between C4 0 of PLP and the a-amino group of the substrate; the electron-withdrawing role of the pyridine ring as an electron sink; and the nucleophilic attack on the a-substituent of an a-carbon or carboxylate group. As is the case in many general PLP enzymes, especially those of other amino acid deaminases, the phAHP can only abstract a-proton from L and Dserine efficiently preceding deamination at high temperature. The active-site structure of the ACC–phAHP complex is very similar to that of the ACChACCD (K51T) complex, whose reaction is compulsively stopped by mutation. The position of the amino group from catalytic Lys54 is far from ACC (Figure 10(a)), whereas, in hACCD, the corresponding lysine residue (Lys51) must be near ACC so that it can play an important role in abstracting a bproton. Presumably, the electron density of the ACC cyclopropane ring influenced by the pyridine ring of PLP through an external aldimine group is different between phAHP and hACCD. The charge density of the pyridine ring is strictly modulated by various factors in the neighborhood of the pyridine nitrogen atom in the PLP enzyme.18–21 The pyridine nitrogen atom of hACCD exists within hydrogen ˚ ) of the side-chain carboxyl bond distance (2.5 A oxygen atom of Glu296, which is not the case for other members of the TRPSb family. The crystallographically analyzed enzymes of this family are TRPSb,22 O-acetylserine sulfhydrylase (cysteine synthase),23 threonine deaminase,24 threonine

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Structural and Enzymatic Properties of ACCD

Figure 10

(legend opposite)

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Structural and Enzymatic Properties of ACCD

synthase,25 and cystathionine b-synthase.26 In these structures, the serine or threonine side-chain is positioned near the pyridine nitrogen atom. In the phAHP structure, the hydroxyl group of Thr308 side-chain approaches the corresponding nitrogen atom (Figure 6). In brief, the circumstances around this region of phAHP are not the same as hACCD, but are the same as those for the other enzymes belonging to the TRPSb family. In a previous paper we demonstrated that the E296Q mutation of hACCD inactivates ACCD activity, and this inertness has been attributed to the difference in the pKa values of Glu and Gln, which influences the electron density of the pyridine ring of PLP. The inertness of phAHP in response to ACC may be similarly explained; the pKa value of threonine is closer to that of glutamine than glutamate. To date, ACC deaminase activity has been reported from only a few organisms. The sequence alignment of the ACCDs including the putative ones (Figure 2) shows that glutamate close to the pyridine nitrogen atom (Glu296 for hACCD) is completely conserved for these genuine ACCDs, whereas it is replaced by threonine in the putative ACCDs (Thr283 for phAHP). Further, the genuine ACCDs have a leucine residue (Leu323 for hACCD) at the position close to the glutamate. This position is also replaced by a second threonine residue in the putative ACCDs (Thr308 for phAHP). In the tertiary structure, the leucine residue (Leu323 for hACCD) provides space for the long side-chain of the glutamate residue by orienting itself in the opposite direction (Figure 6(b)). Thus, it could be concluded that pairwise mutation for these two positions is necessary for recovering this “residual transposes” and acquiring the ACCD activity. We predict that among the putatively annotated ACCDs, those that have threonine residues at the corresponding positions will be unable to induce ACCD activity.

Materials and Methods Cloning, expression and purification The phAHP gene (ORF identification number PH0054) was amplified by PCR using the oligonucleotide primers 5 0 -G GTG GTG CAT ATG CAT CCG AAG ATC TTT GC-3 0 (underlining indicates the upper primer containing the NdeI site) and 5 0 GG AAG AAA AGG GAA TTC CTA AAG TAG TGA C-3 0 (underlining indicates the lower primer containing the EcoRI site). The PCR products were ligated into a pGEM-T Easy vector (Promega) and the nucleotide sequences were confirmed by DNA

sequencing using an Applied Biosystems dRhodamine kit. The phAHP genes digested by NdeI and EcoRI from the pGEM-T Easy vector were introduced into a pET-22b(C) vector (Novagen). After transformation in E. coli BL21(DE3) (Stratagene), the cells were grown at 80 8C in LB medium containing 50 mg mlK1 ampicillin until A600 nm reached 0.8–1.0. The pyridoxine (salt with hydroxyl chloride) was added to 10K4 M so that PLP was easily synthesized in the host cell. Over-expression of the recombinant phAHP was induced by 1 mM isopropylthio- D -galactoside (IPTG). After IPTG injection, the cells were grown for seven hours with constant shaking, and were harvested by centrifugation at 4000 rpm for 15 minutes. The cells were then suspended in buffer A (40 mM potassium phosphate (pH 6.5)) and homogenized twice by a French press (Amico Inc.) at 1000 lb/in2 (1 lb/in2z6.9 kPa). After centrifugation at 40,000 rpm for one hour, the supernatant was incubated at 80 8C for 30 minutes then centrifuged at 20,000 rpm for 30 minutes. The supernatant was applied to an anion-exchange DEAE-Sepharose (Amersham Biotech) column equilibrated with buffer A. After washing with buffer A, the bound protein was eluted using a linear gradient of 0–0.5 M KCl added to buffer A. The fractions containing phAHP were then applied to HiLoad 20/60 Superdex-200 columns (Amersham Biotech) equilibrated with buffer B (40 mM potassium phosphate buffer (pH 6.5), 200 mM KCl, 1 mM EDTA, 1 mM DTT, 10K5 M PLP). The phAHP was collected as a single peak. The purity of the protein was analyzed by SDSPAGE and MALDI-TOF mass spectrometry (Voyager DE PRO; PerSeptive Biosystems). Approximately 10–15 mg mlK1 of phAHP was obtained from one liter of culture. Selenomethionyl-substituted phAHP was expressed in E. coli cells B834 (DE3) (Novagen) in M9 minimum medium containing 25 mg ml K1 of Se-Met, 50 mg mlK1 of ampicillin, and 35 mg mlK1 of chloramphenicol. Se-Met phAHP was overexpressed and purified by almost the same method as that used with native phAHP. Measurement of deaminase activity The deaminase activity was measured toward several amino acids, including ACC, D-Ala, L-Ala, DSer, and L-Ser, as described.11 After 100 ml of protein solution had been incubated at each reaction temperature for three minutes, 0.1 M substrate containing 0.1 M Tris, 0.045 M KH2PO4(pH 8.5) was added, then the solution was incubated for

Figure 10. Comparison of active sites between phAHP and hACCD. (a) Superimposition of ACC complexed phAHP (yellow) and ACC complexed hACCD (K51T, green). (b) Stereo view showing the active site of phAHP. The main chain is colored blue and the side-chains that form the pathway from the solvent region are colored gray. PLP is shown in yellow ball-and-stick model. The black arrow indicates the plausible substrate pathway. (c) Stereo view showing the active site of hACCD. The main-chain is colored green and the side-chains that form the cavity from the solvent region are colored gray. PLP is shown in yellow ball-and-stick model. The orientation is the same as that in (b).

1010 10–15 minutes at each reaction temperature. The amounts of compounds that included a keto-group were detected by hydrazone formation with 2,4dinitrophenylhydrazine according to the description by Hatfield et al.27 A unit was defined as the amount of enzyme required to form 1 mmol of aketobutylate in one minute. Specific activities are expressed in units/mg protein. Identification of the reaction product After the addition of 1.0 M HCl (5 ml) to the above reaction solution, the resulting mixture was extracted with ethyl/acetate (EtOAc) (6!40 ml). The EtOAc layers were dried over MgSO4 and concentrated in vacuo. The EtOAc extract solution in 0.5 M HCl/acetone (8 : 2, v/v; 100 ml) was subjected to analysis without further purification. The GC–MS analysis was performed using a fused silica capillary column (CBP20; 0.22 mm! 25 m, Shimadzu) with a HP5890 gas chromatograph and a JMS-600H mass spectrometer (JEOL). An aliquot (0.6 ml) of the sample solution was injected at the injection port temperature of 200 8C. The column temperature was maintained at 120 8C for one minute followed by heating to 140 8C at increments of 10 deg. C/minute. In this step the solvent was passed. Then the temperature was held at 140 8C for one minute then increased to 160 8C at 1 deg. C/minute. A total ion chromatogram of the sample showed a peak whose mass spectrum exhibited the molecular ion at m/z 88. It was identified as pyruvic acid by comparison with a result in the GC–MS analysis of sodium pyruvate (aqueous HCl solution). Further confirmation was obtained by following the decrease in absorbance at 340 nm resulting from the oxidation of NADH (3340Z6.22 mMK1 cmK1) to NAD in the presence of lactate dehydrogenase, which catalyzes the reduction of pyruvate formed by phAHP. Each mixture (1 ml) of L and D-serine (50 mM) with phAHP (1 mg) was reacted at 80 8C for six hours. Each mixture (30 ml) was added into a cuvette containing 50 mM potassium phosphate (pH 7.0), 0.5 mM NADH, and then reacted for an additional one minute after addition of lactate dehydrogenase at 30 8C (0.65 unit/ml) in a total volume of 100 ml. The oxidation of NADH was followed at 340 nm at 30 8C on a DU series 600 spectrometer (Beckman), and the light-path of the cuvette was 1 cm. Crystallization and data collection After purification, native and Se-Met phAHPs were dialyzed overnight at 277 K in buffers C (10 mM potassium phosphate (pH 7.5)) and D (0.1 M potassium phosphate (pH 7.0)) respectively, and concentrated to 5 mg mlK1 using Apollo 7 ml (Orbital Biosciences). Crystals were grown under the conditions of 0.1 M sodium/potassium phosphate (pH 6.2–7.0), 2.0 M ammonium sulfate, and 3%–8% (v/v) 2-propanol using the hanging-drop,

Structural and Enzymatic Properties of ACCD

vapor-diffusion method by mixing 2.5 ml of protein sample with 2.5 ml of reservoir solution. Crystals grew up to 0.4 mm!0.4 mm!0.1 mm in a week at 18 8C. For crystallization of the phAHP–ACC complex, protein solution dialyzed in buffer C was concentrated to 10 mg mlK1 and mixed with an equal volume of 0.1 M ACC solution (0.1 M Tris, 0.045 M KH2PO4 (pH8.5)). The complex crystals were obtained under the condition of 0.1 M NaHepes buffer (pH 7.5), 10% (w/v) 2-propanol and 13–15% (w/v) polyethylene glycol 4000. Se-MAD data were collected at beamline BL40B2 of SPring-8 using the ADSC CCD detector (quantum 4D). Three wavelengths were selected for the data collection based on the fluorescence spectrum for the Se K edge, corresponding to the ˚ , peak), the minimum f 00 maximum f 0 (0.9792 A ˚ ˚, (0.9795 A, edge), and a reference point (0.9640 A remote). The native and complex data collections were carried out at beamline BL41XU of SPring-8 using the MAR CCD detector with wavelengths of ˚ and 1.0000 A ˚ , respectively. All data sets 0.9700 A were collected at 100 K after crystals were soaked into anti-freezing buffer containing 15–20% (v/v) glycerol. The crystals of native and Se-Met belong to the space group P3221 with unit cell parameters of ˚ , cZ115.0 A ˚ , and gZ1208, and there aZbZ122.3 A are three molecules in an asymmetric unit (VM28Z ˚ 3/Da; VsolvZ53.9%), whereas the crystals of 2.69 A the ACC complex belong to space group P1 with ˚ , bZ147.2 A ˚ , cZ unit cell parameters of aZ105.8 A ˚ , aZ73.18, bZ90.18, and gZ68.48, and 24 149.0 A molecules are included in an asymmetric cell (VMZ 2.43 A3/Da; VsolvZ49.0%). All reflection data were integrated and scaled using MOSFLM 29 and SCALA.30 The statistics of data collection and processing are shown in Table 2. Structure determination and refinement The structure of phAHP was determined using the multi-wavelength anomalous diffraction (MAD) method. Three out of 12 Se sites were determined by program SHELX-9731 and used to calculate the ˚ resolution. The other eight Se initial phases at 2.8 A sites were found by difference Fourier methods and used to re-phase by program SHARP.32 The noncrystallographic symmetry (NCS) matrices were obtained from Se sites. The density modification by NCS averaging with phase extension procedure to ˚ resolution using native data was carried out 2.5 A by program SOLOMON.33 The initial model was built using program O.34 The phasing statistics are summarized in Table 3. The structure of the phAHP–ACC complex was determined by the molecular replacement method using the dimer of phAHP as a search model. The orientations and positions of 11 dimer molecules in the unit cell were determined with program AMoRe.35 In this step, the R value was 43.2%. After the phase improvement by NCS averaging using dm,36 the electron density map clearly showed 12 dimer molecules.

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Structural and Enzymatic Properties of ACCD

Table 2. Data statistics Native

ACC complex

BL41XU 0.9700 P3221 ˚ aZbZ122.3 A ˚ cZ115.0 A gZ1208 39–2.5 (2.64–2.5) 34,161 (4953) 2668 (282) 98.6 (98.6) 6.0 (6.0) 7.9 (2.9) 7.3 (28.5)

BL41XU 1.0000 P1 ˚ aZ105.8 A ˚ bZ147.2 A ˚ cZ149.0 A aZ73.18, bZ90.18 gZ68.48 39.1–2.60 (2.74–2.60) 572,213 (87,298) 244,791 (35,772) 100.0 (100.0) 2.3 (2.4) 7.2 (2.2) 6.4 (36.7)

7452 94 60 20–2.5 20.12 26.95 55.29

59,616 970 578 10–2.7 29.18 32.0 52.69

0.0097 1.542 23.01 1.026

0.020 1.973 22.96 1.168

A. Data collection Beam line ˚) Wavelength (A Space group Cell parameters

˚) Resolution range (A Observed reflections Unique reflections Completeness (%) Multiplicity Averaged I/s(I) Rmeasa (%) Rl b (%) B. Refinement No. non-hydrogen atoms Protein Water Other ˚) Resolution range (A Rwork (%)c Rfree (%)d ˚ 2) Average B-factor (A r.m.s.d. from ideality ˚) Bond lengths (A Bond angles (deg.) Dihedral angles (deg.) Improper angles (deg.)

Peak

0.9792

34–2.6 (2.74–2.6) 30,569 (4386) 2754 (270) 99.9 (99.9) 9.8 (9.0) 11.1 (2.7) 7.0 (31.4) 6.9 (12.4)

Se-Met edge BL40B2 0.9795 P3221 ˚ aZbZ122.0 A ˚ cZ114.2 A gZ1208 34–2.6 (2.74–2.6) 30,676 (4442) 2772 (280) 99.9 (99.9) 10.8 (10.8) 9.5 (2.8) 6.6 (28.7) 7.2 (14.6)

Remote

0.9640

34–2.8 (2.95–2.8) 24,646 (3537) 2394 (234) 99.9 (99.9) 10.2 (9.5) 11.6 (3.1) 6.4 (26.7) –

Values in parentheses refer to data in the highest resolution shell in each data set. P P P P a Rmeas Z h ½ðmK 1Þ=m
1=2 j j! IOh KIh;j j= h j Ih;j , where hIih is the mean intensities of symmetry-equivalent reflections and m is redundancy. P P b Rl Z hP jFli K Fl0 j= h jF Pl0 j, where l0 is reference (remote) data. c Rwork Z h jjFo jK jFc jj= h jFo j, where Fo and Fc are the observed and calculated structure-factor amplitudes. d Rfree was calculated for Rwork, using a random 8% test set of reflections.

The models of both the phAHP and phAHP– ACC complexes were refined using the program CNS 1.1.37 The NCS restraints were used during the refinement. Eight percent of the reflection data were set aside for the calculation of the free R-factor to

Se-Met crystal

Peak 0.5457 1.989 3.091

Atomic coordinates The atomic coordinates of native and ACC-bound phAHP have been deposited in the RCSB Protein Data Bank (accession codes 1J0A and 1J0B).

Table 3. Phasing statistics

˚) Data (15–2.8 A Space group MAD phasing Rcullis_isoa Phasing power_isob Phasing power_anoc FOMd FOM after NCS averaging

monitor the refinement. The structure refinement statistics are also shown in Table 2.

˚ 15–2.8 A P3221 Edge 0.5592 1.991 1.649

Remote – 2.504 0.6516 0.9593 ˚) (15–2.5 A

a Rcullis_iso is the mean residual lack of closure error divided by dispersive difference. Values are for centric reflections. b Phasing power_iso is the root-mean-square of FH/E, where FH is the dispersive difference of FH and E is the lack of closure error. c Phasing power_ano is as for phasing_power_iso except that FH is the anomalous difference of FH. d FOM, mean figure-of-merit. FOM after the NCS averaging with phase extension was calculated with SOLOMON.33

Acknowledgements We thank H. Oikawa and T. Mie (Graduate School of Agriculture, Hokkaido University) for synthesizing ACC. We also thank K. Miura and M. Kawamoto of the Japan Synchrotron Radiation Research Institute (JASRI) for their kind help in data collection on beamline BL40B2 and BL41XU at SPring-8. This work was supported by the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Sports and Culture of Japan.

1012

References 1. Sheehy, R. E., Honma, M., Yamada, M., Sasaki, T., Martineau, B. & Hiatt, W. R. (1991). Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase. J. Bacteriol. 173, 5260–5265. 2. Klee, H. J., Hayford, M. B., Kretzmer, K. A., Barry, G. F. & Kishore, G. M. (1991). Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants. Plant Cell, 3, 1187–1193. 3. Jacobson, C. B., Pasternak, J. J. & Glick, B. R. (1994). Partial purification and characterization of ACC deaminase from the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2. Can. J. Microbiol. 40, 1019–1025. 4. Campbell, B. G. & Thomson, J. A. (1996). 1-Aminocyclopropane-1-carboxylate deaminase genes from Pseudomonas strains. FEMS Microbiol. Letters, 138, 207–210. 5. Shah, S., Li, J., Moffatt, B. A. & Glick, B. R. (1998). Isolation and characterization of ACC deaminase genes from two different plant growth-promoting rhizobacteria. Can. J. Microbiol. 44, 833–843. 6. Minami, R., Uchiyama, K., Murakami, T., Kawai, J., Mikami, K. Yamada, T. et al. (1998). Properties, sequence, and synthesis in Escherichia coli of 1aminocyclopropane-1-carboxylate deaminase from Hansenula saturnus. J. Biochem. (Tokyo), 123, 1112–1118. 7. Jia, Y. J., Kakuta, Y., Sugawara, M., Igarashi, T., Oki, N., Kisaki, M. Shoji, T. et al. (1999). Synthesis and degradation of 1-aminocyclopropane-1-carboxylic acid by Penicillium citrinum. Biosci. Biotechnol. Biochem. 63, 542–549. 8. Yao, M., Ose, T., Sugimoto, H., Horiuchi, A., Nakagawa, A. Wakatsuki, S. et al. (2000). Crystal structure of 1-aminocyclopropane-1-carboxylate deaminase from Hansenula saturnus. J. Biol. Chem. 275, 34557–34565. 9. Ose, T., Fujino, A., Yao, M., Watanabe, N., Honma, M. & Tanaka, I. (2003). Reaction intermediate structures of 1-aminocyclopropane-1-carboxylate deaminase. J. Biol. Chem. 278, 41069–41076. 10. Adams, D. O. & Yang, S. F. (1979). Ethylene biosynthesis: identification of 1-aminocyclopropane1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl Acad. Sci. USA, 76, 170–174. 11. Honma, M. & Shimomura, T. (1978). Metabolism of 1aminocyclopropane-1-carboxylic acid. Agric. Biol. Chem. 42, 1825–1831. 12. Glick, B. R., Jacobson, C. B., Schwarze, M. M. K. & Pasternak, J. J. (1994). 1-Aminocyclopropane-1-carboxylic acid deaminase mutants of the plant growth promoting rhizobacterium Pseudomonas putida GR122 do not stimulate canola root elongation. Can. J. Microbiol. 40, 911–915. 13. Glick, B. R., Penrose, D. M. & Li, J. (1998). A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria. J. Theor. Biol. 190, 63–68. 14. Kawarabayasi, Y., Sawada, M., Horikawa, H., Haikawa, Y., Hino, Y. Yamamoto, S. et al. (1998). Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. DNA Res. 5, 55–76. 15. Kabsch, W. & Sander, C. (1983). Dictionary of protein secondary structure: pattern recognition of hydrogenbonded and geometrical features. Biopolymers, 22, 2577–2637.

Structural and Enzymatic Properties of ACCD

16. Shaw, J. P., Petsko, G. A. & Ringe, D. (1997). Determination of the structure of alanine racemase ˚ resolution. from Bacillus stearothermophilus at 1.9-A Biochemistry, 36, 1329–1342. 17. Collaborative Computational Project Number 4. (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallog. sect. D, 50, 760–763. 18. Clark, P. A., Jansonius, J. N. & Mehler, E. L. (1993). Semiempirical calculations of the electronic absorption spectrum of mitochondrial aspartate aminotransferase. 2. The 2-methylaspartate complex. J. Am. Chem. Soc. 115, 9789–9793. 19. Clark, P. A., Jansonius, J. N. & Mehler, E. L. (1993). Semiempirical calculations of the electronic absorption spectra of vitamin B6 derivatives in the active site of mitochondrial aspartate aminotransferase. J. Am. Chem. Soc. 115, 1894–1902. 20. Jhee, K. H., McPhie, P., Ro, H. S. & Miles, E. W. (1998). Tryptophan synthase mutations that alter cofactor chemistry lead to mechanism-based inactivation. Biochemistry, 37, 14591–14604. 21. Jhee, K. H., Yang, L. H., Ahmed, S. A., McPhie, P., Rowlett, R. & Miles, E. W. (1998). Mutation of an active site residue of tryptophan synthase (beta-serine 377) alters cofactor chemistry. J. Biol. Chem. 273, 11417–11422. 22. Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W. & Davies, D. R. (1988). Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium. J. Biol. Chem. 263, 17857–17871. 23. Burkhard, P., Rao, G. S., Hohenester, E., Schnackerz, K. D., Cook, P. F. & Jansonius, J. N. (1998). Threedimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium. J. Mol. Biol. 283, 121–133. 24. Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chinchilla, D. & Eisenstein, E. (1998). Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase. Structure, 6, 465–475. 25. Thomazeau, K., Curien, G., Dumas, R. & Biou, V. (2001). Crystal structure of threonine synthase from Arabidopsis thaliana. Protein Sci. 10, 638–648. 26. Meier, M., Janosik, M., Kery, V., Kraus, J. P. & Burkhard, P. (2001). Structure of human cystathionine beta-synthase: a unique pyridoxal 5 0 -phosphatedependent heme protein. EMBO J. 20, 3910–3916. 27. Hatfield, G.W. & Umbarger, H. E. (1971) L-Threonine deaminase, biosynthetic In Methods in Enzymology (Tabor, M. & Tabor, C. W., eds), vol. 17(B), pp. 561–566, Academic Press, New York. 28. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491–497. 29. Leslie, A. G. W. (1993). Auto-indexing of rotation diffraction images and parameter refinement. In Proceedings of the CCP4 Study Weekend on Data Collection and Processing (Sawyer, L., Isaacs, N. & Bailey, S., eds), Daresbury Laboratory, Warrington. 30. Evans, P. R. (1993). Data reduction. In Proceedings of the CCP4 Study Weekend on Data Collection and Processing (Sawyer, L., Isaacs, N. & Bailey, S., eds), pp. 114–122, Daresbury Laboratory, Warrington. 31. Sheldrick, G. M., Dauter, Z., Wilson, K. S., Hope, H. & Sieker, L. C. (1993). The application of direct methods and Patterson interpretation to high-resolution native protein data. Acta Crystallog. sect. D, 49, 18–23. 32. de La Fortelle, E. & Bricogne, G. (1997). Maximumlikelihood heavy-atom parameter refinement for

Structural and Enzymatic Properties of ACCD

multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 294–472. 33. Abrahams, J. P. & Leslie, A. G. W. (1996). Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystllog. sect. D, 52, 30–42. 34. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallog. sect. A, 47, 110–119.

1013 35. Navaza, J. (1994). AMoRe: an automated package for molecular replacement. Acta Crystallog. sect. A, 50, 157–163. 36. Cowtan, K. (1994). dm: an automated procedure for phase improvement by density modification. Joint CCP4 ESF-EACBM Newsletter Protein Crystallog. 31, 34–38. 37. Bru¨nger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P. Grosse-Kunstleve, R. W. et al. (1998). Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallog. sect. D, 54, 905–921.

Edited by D. Rees (Received 17 February 2004; received in revised form 10 June 2004; accepted 11 June 2004)