Two Structures of Alliinase from Alliium sativum L.: Apo Form and Ternary Complex with Aminoacrylate Reaction Intermediate Covalently Bound to the PLP Cofactor

doi:10.1016/j.jmb.2006.11.041

J. Mol. Biol. (2007) 366, 611–625

Two Structures of Alliinase from Alliium sativum L.: Apo Form and Ternary Complex with Aminoacrylate Reaction Intermediate Covalently Bound to the PLP Cofactor Linda J.W. Shimon 1 ⁎, Aharon Rabinkov 2 , Irina Shin 2 , Talia Miron 2 David Mirelman 2 , Meir Wilchek, 2 and Felix Frolow 3 ⁎ 1

Department of Chemical Research Support, The Weizmann Institute of Science, Rehovot 76100, Israel 2

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel 3

Department of Molecular Microbiology and Biotechnology and The Daniella Rich Institute for Structural Biology, Tel Aviv University, Ramat Aviv 69978, Israel

Alliinase (alliin lyase EC 4.4.1.4), a PLP-dependent α, β-eliminating lyase, constitutes one of the major protein components of garlic (Alliium sativum L.) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid alliin ((+S)-allyl-L-cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl-Lcysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo-enzyme was crystallized in tetragonal form. These structures of alliinase with associated glycans were resolved to 1.4 Å and 1.61 Å by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to “short chain complex vacuole type” oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the ε-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo-form is nearly identical to that seen in the holo-form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor. © 2006 Elsevier Ltd. All rights reserved.

*Corresponding authors

Keywords: alliinase; X-ray structure; pyridoxal 5'-phosphate aminoacrylate; plant enzyme glycosylation

Introduction Garlic (Alliium sativum L.) is a plant grown throughout the world that is valued both as a food and as a folk-medicine. For generations, garlic has been known for its remarkable medicinal properties, which include antibiotic as well as hypolipidemic, antithrombotic, antihypertensive and anticancer E-mail addresses of the corresponding authors: [email protected]; [email protected]

activities.1 The pungent and unmistakable smell of garlic is a result of the reaction of the enzyme alliinase (E.C. 4.4.1.4) and its substrate, the nonprotein amino acid, alliin.2 Under normal circumstances, in the intact cells of the garlic bulbs, these two molecules are physically separated: the enzyme alliinase is compartmentalized in the vacuoles while the alliin is localized in the cell cytoplasm.3 However, as has been commonly observed, when the garlic bulbs are crushed or injured, the enzyme and its substrate are brought into contact so that the reaction shown in Scheme 1 can take place.

0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.

612

Two Structures of Alliinase from Alliium sativum L.

Scheme 1. Alliinase reaction with alliin: the key intermediates in the reaction pathway are labeled. In its resting state, in the absence of substrate, the enzyme forms a covalent, Schiff base with Lys251, the internal aldimine (e). When the substrate alliin (c) is present, it binds covalently to the PLP to form the external aldimine (d). The C-S bond is cleaved resulting in the release of allylsulfenic acid (a). Two allylsufenic acid molecules join to form allicin (b) and leaving aminoacrylate (f) bound to the PLP. In the final step, this aminoacrylate (f) is lysed from the PLP and hydrolyzed, yielding pyruvate and ammonia (g). The PLP is then free to reform the internal aldimine with Lys251 (e).

The reaction catalyzed by alliinase (Cys sulfoxide lyase, alliin lyase, C-S-lyase), which requires pyridoxal-5′-phosphate (vitamin B6 phosphorylated derivative-PLP) as a cofactor, is categorized as α,βelimination reaction and involves an aminoacryl intermediate bound to PLP (Scheme 1). The enzyme converts the substrate alliin ((+)-S-allyl-L-cysteine sulfoxide) to allylsulfenic acid and aminoacrylate. Two molecules of allylsulfenic acid condense spontaneously to form the sulfur-containing volatile, allicin (diallylthiosulfinate). The aminoacrylate is lysed from the PLP and spontaneously decomposes to pyruvate and ammonia, thereby restoring the internal aldimine as the original state of the enzyme, ready for the next reaction cycle. Allicin is the parent compound for a majority of other sulfur-containing biologically active metabolites in the garlic such as the thiosulfinates, allyl sulfides, dithiines and ajoenes4,5 that are produced through sub-sequential non-enzymatic reactions. Most of garlic's beneficial health effects are ascribed to these sulfur-containing compounds. Alliinase from garlic cloves (A. sativum) has been isolated and is well characterized. It has been shown to be a dimer with two equal subunits each of 448 amino acid residues with a total molecular mass of 103 kDa. 6 Alliinase belongs to the family of mannose-rich glycoproteins with an estimated carbohydrate content 6, 7 of about 5.5–6%. The enzyme was found to form a stable complex with

a mannose specific lectin8 ASA-1. The pH optimum of alliinase activity is 6.5 and its isoelectric point was determined6 between 6.0 and 7.0. Alliinase from other Allium genus plants such as A. sepa (onion) bulb9 and roots,10 A. tuberosum (Chinese chives),11 A. ursinum (ramson or wild garlic)12 and A. porrum (leek)13 have been identified, the sequence homology between these proteins varies from 51 to 70 %. The distinctive odors and flavors of the Alliium family members are presumed to be due to the different patterns of accumulation of the alliin and/or the distinct substrate.14 In addition, alliinases from roots may differ significantly from those located in the bulb (A. sativum and A. sepa).6,10 Alliinase enzymes have also been identified in Acacia farnesiana seedling, Brassica oleracea buds, bacteria and fungi.15–19 Alliinase activity is dependent upon the PLP cofactor.20 PLP-dependent proteins play pivotal role in the assimilation and metabolic transformations of nitrogen and sulfur-containing compounds, performing a broad variety of elimination, exchange, and condensation reactions (e.g. transamination, racemization, decarboxylation, cleavage or elongation of carbon chains, elimination or replacement of substituents), particularly at the α, β and γ C-atoms in amino acids, and in other NH2-containing compounds.21 Several enzymes are known to catalyze α,β-elimination reaction with S-substituted cysteine residues (cysteine S-conjugates) and yield pyruvate,

613

Two Structures of Alliinase from Alliium sativum L.

ammonia and RSH.22 This group of PLP-dependent enzymes are involved in the metabolic pathways of amino acids such as cysteine, homocysteine, methionine and their derivatives. The C-S lyases belong to the fold type I group of the PLP-dependent enzymes,23 a group that contains aspartate aminotransferase as its most representative member. The overall sequence similarity of alliinases with other C-S lyases is low (only about 20%). Alliinase from garlic has been previously crystallized24,25 and its structure has been reported.26 Here we present two high resolution structures of alliinase from garlic, one in the complex with its reaction intermediate, aminoacrylate covalently bound to the PLP cofactor (1.40 Å) and the second one, intentionally produced apo-form of the enzyme (1.60 Å). These structures are compared with internal aldimine form of PLP from the previously reported structure of alliinase 26 and with the structure of aminoacrylate complex of PLP with cystine C-S lyase from Synechocystis,27 one of only two other aminoacrylate structures reported to date in the Protein Data Bank.

Results and Discussion Structure determination The structure of monoclinic holo-form and tetragonal apo-forms of alliinase were determined by Xray crystallography to 1.40 Å and 1.61 Å resolution, respectively, using molecular replacement with previously determined alliinase structure26 (PDB code 1LK9) as a search model and refined to an R factor of 16.9% (Rfree 20.5%) and R = 16.4% (Rfree = 17.9%). A summary of refinement statistics is given in Table 1. The refined crystal structure of the holo-form shows well-defined electron density for residues 1– 425 in monomers A, B, C and D. The final model shows electron density for a total of 26 saccharide groups attached (per asymmetric unit containing four monomers) in ten different N-linked glycan chains: 17 N-acetylglucosamines (GlcNAc), four mannose (Man), four fucose (Fuc) and one xylose (Xyl). The refined structure of the apo-form has only one molecule per asymmetric unit and a total of seven saccharide groups. In the holo-form, there are two PLP cofactor molecules per alliinase dimer for which there is unambiguously defined electron density (see below); the structure of the cofactor can be resolved and reveals the presence of an aminoacrylate adduct (henceforth referred to in text as PLP-AA). In addition 2197 water molecules and four chloride ions (one per monomer) have been resolved. In the apo-form the PLP cofactor is not seen. Monomer structure Monomers of the holo and apo forms of alliinase are very similar with an rms difference of 0.27 Å.

Table 1. Crystal data and refinement statistics

Crystal data Space group Temperature Unit-cell parameters Internal scaling Resolution (Å) Reflections measured Unique reflections Completeness (%) I/σ(I) (average) Rsym Model refinement Resolution range (Å) Molecules (AU) No. protein residues No. cofactor molecules No. sugar rings No. non-H atoms No. solvent molecules No. of Cl atoms Wilson B factor (Å2) Overall B factor (Å2) PLP averaged B factor (Å2) Rcryst Rfree 5% of data Geometry r.m.s. bonds (Å) r.m.s. bonds angles (°) r.m.s. planar groups (°)

Monoclinic-holo

Tetragonal-apo

P21 100 K a = 67.67 Å, b = 126.89 Å, c = 102.66 Å, β = 97.30°

P41212 100 K a = b = 80.96 Å, c = 163.69 Å

46.0–1.40 1,008,127

42.0–1.60 570,191

327,314

72,527

96.5(73.62)

99.7(100.0)

40.4 0.049

29.2 0.089

46.0–1.40(1.43–1.40)

42.0–1.60(1.63–1.60)

4 1704

1 425

4

0

26 16,330

7 4134

2197

496

4 17.90

1 22.07

18.43

25.47

19.43

N/A

0.169 0.205

0.164 0.179

0.014 1.503

0.010 1.345

0.009

0.005

(monomer A of the holo form is used for the following comparisons). The fold of alliinase (Figure 1) is related to that of other PLP-dependent enzymes of the aminotransferase family.28 The monomer of alliinase has triangular prism shape with overall dimensions 60 Å × 60 Å × 60 Å × 40 Å and consists of two major domains (Figure 1): the smaller domain, comprising the N and C termini regions residues 1–100 and 311–425 (domain 1) is the substrate binding domain, and residues 101–310 is the larger, central PLP binding domain (domain 2 contains six cysteine residues, which form three disulfide bonds). Disulfide bridge Cys368–Cys376 is located close to the C terminus end of the C-terminal domain and through series of interactions is supporting PLP binding site. The PLP-binding domain 2 (101–310) has a αβα topology, a classic domain fold type 1 observed in aminotransferases29 and some other PLP-dependent enzymes.

614

Figure 1. Ribbon diagram of the alliinase dimer, AB. Monomer A is shown in red, monomer B in blue. The small, substrate-binding domains are shown in dark tones, and the PLP binding domains are shown lighter. The cofactor intermediate PLP-AA is shown as spheres and colored according to element. The N and C termini are marked. Prepared with PyMOL.70

Dimer structure: monoclinic holo-form The alliinase molecule is known to be a homodimer.30 Two dimers of approximate dimensions 52 Å × 65 Å × 100 Å each, denoted AB and CD (Figure 1), are found in the asymmetric part of the unit cell. The monomers are related by a noncrystallographic 2-fold symmetry, with r.m.s. differences between the monomers in AB and CD of 0.25 Å and 0.23 Å, respectively and dimer to dimer r.m.s. differences of 0.34 Å (r.m.s. differences calculated based on Cα atoms, fitting was performed using the McLachlan algorithm31 as implemented in the program ProFit (Martin, A.C.R.†)). Extensive contacts occur at the interface between the monomers. The area of monomer–monomer interface that is excluded upon dimerization is 4770 Å, which corresponds to 21% of the total solvent accessible surface area32 (Protein-Protein Interaction server‡). The alliinase molecule is biologically active as a dimer with the active sites built by residues from both monomers. There are two identical PLP binding sites per dimer and they are located at the dimer interface so that both monomers are involved in the binding of each cofactor molecule. The two PLP binding sites in the dimer are positioned approximately 34 Å apart. PLP cofactor entrance channel axes are oriented in the plane, perpendicular to the non-crystallographic 2-fold axis of the dimer, bringing them to antiparallel orientation (Figure 1). The otherwise very precise 2-fold relation between monomers in dimers is broken by systematic deviations from symmetry at various sites. The con† http://www.bioinf.org.uk/software/profit/ ‡ http://www.biochem.ucl.ac.uk/bsm/PP/server/

Two Structures of Alliinase from Alliium sativum L.

formations of some residues on the dimerization interface close to the non-crystallographic 2-fold axis deviate from the symmetry. The most prominent deviations are in CD dimer interface at residues Met2C and Met2D. The main chain atoms in these residues maintain a symmetrical arrangement, and the deviations from symmetry are a result of differences in the side-chain conformations. Met2C extends toward the 2-fold axis and Met2D points away from it. This deviation from the symmetry averts the stereo-chemical clash that would occur if precise symmetry were maintained. The methionine arrangement at the AB dimer interface is different. The very clear electron density map shows no indication of alternate side-chain conformations and exhibits precise symmetry arrangement of the side-chains that brings the Cα atoms of Met2A and Met2B to an energetically impossible 1.0 Å distance. Formation of mixed dimers of the mutually exclusive isoforms of the enzyme may explain this observation. Dimer structure: tetragonal apo-form The tetragonal form of apo-alliinase has one monomer per asymmetric unit; the dimer is formed by strict crystallographic 2-fold axis, thereby exhibiting a fully symmetric interface. The r.m.s. diffrence of the apo and holo dimers is 0.365 Å, which is equivalent to that of two different noncrystallographic dimers in the holo alliinase. The essentials of the dimer interactions with the cofactor binding are preserved. The dimer interface interactions are preserved with only minor differences related to the orientation of the interacting elements. The active site Holo-form active site The active site Lys251 is known to form an internal aldimine (Scheme 1) with the PLP cofactor. However, in the present structure an unexpected state of the enzymatic catalytic active site, in the form of a stable aminoacrylate-PLP complex, has been observed. The electron density in the region of the PLP cofactors is of exceptional quality (Figure 2(a)) making possible an unambiguous identification of the molecule as the aminoacrylate-PLP intermediate as opposed to free PLP cofactor or a ketamine intermediate. The average temperature factor of the four PLP-AA molecules (one per asymmetric unit) is comparable to that of the surrounding protein (see Table 1). There is a distinct separation in the electron density between the aminoacrylate adduct and Lys251 making distance of 3.44 Å between ε-amino group and C4′ (Figure 2(a) and (b)), ruling out detectable presence of the expected internal aldimine bond between the ε-amino group of Lys251 and the aldehyde of the PLP-AA. Instead Lys251 makes a 2.92 Å hydrogen bond interaction with Cβ atom of the aminoacrylate, which resembles the

Two Structures of Alliinase from Alliium sativum L.

615

Figure 2. (a) Stereo plot of the final |2Fo–Fc| electron density maps contoured at 1σ, in the immediate vicinity of the PLP-AA molecule and the surrounding protein region. The molecules are colored by atom type. The position of Lys251 is labeled. Prepared with BOBSCRIPT.71,72 (b) The catalytic pocket and the PLP-AA intermediate. Hydrogen bonds to the interacting residues of the protein and to the relevant water molecules are shown as blue dotted lines. Prepared with MOLSCRIPT.73

initial stage of Michael addition formation (see Eliot & Kirsch review and references therein33). In the present structure Lys251 (and side-chain rotamers of Lys251) cannot approach C4′ of PLP to make a covalent bond without a substantial rearrangement of the secondary structure. The unexpected state of the active site described above is maintained in all four independent monomers of the asymmetric unit. The active site of alliinase is located near the center of the monomer–monomer interface The PLP-AA intermediate sits within a buried catalytic pocket that is formed at the dimer interface and is tightly bound to the protein by a large number of specific interactions to the aminoacrylate moiety (AA) of the PLP-AA. The Cα-CH2 bond length of aminoacrylate is 1.31 Å, indicative of a short alkene bond. The AA moiety methylene carbon donates its two hydrogen atoms, forming hydrogen bonds of 2.91 Å with the main chain O of Gly64 and of 2.91 Å to the ε-amino

group of Lys251. The carboxylate group of the AA forms a salt-bridged dimer with the guanidinium group of Arg401 and two hydrogen bonds of 2.95 Å and 2.79 Å are formed (Figure 2(b)). A bond of 2.92 Å to the main chain N of Gly64 additionally buttresses one of the carboxylate group oxygen atoms. Both of these residues, Gly64 and Arg401, interacting with AA moiety come from the substrate-binding domain. A number of cofactor-enzyme hydrogen bonds are also formed with the PLP moiety of the PLP-AA. The O3 group forms bonds of 2.8 Å to residue Asn207 ND2 and 2.6 Å to the OH group of Tyr228. In addition Asn207 Oδ1 contacts NH1 of Arg401 and may transmit to the latter information about changes in the orientation of the PLP pyridine ring. The N1 pyridine nitrogen of the hetero-cycle is bound with a distance of 2.65 Å to Asp225 OD2. In type 1, PLPdependent enzymes, Asp225 is the most structurally

616 and functionally conserved residue. The phenol ring of Tyr165 is positioned 3.9 Å above the pyridine ring of the PLP-AA, further stabilizing the cofactor through ring stacking interactions (Figure 2(b)). Interactions between the phosphate group and the enzyme anchor the cofactor to both subunits of the dimer. A total of eight hydrogen bonds are formed between the phosphate oxygen atoms of PLP-AA and the protein residues Val132, Thr133, Thr248, Ser250, Arg259, which belong to the same monomer and Tyr92 which is donated by the second monomer in a dimer. No water molecules form direct interactions with the phosphate oxygen atoms of PLP-AA, unlike in many other PLP-dependent enzymes.34 Active site water molecules in holo structure There are several water molecules positioned relatively closely to aminoacrylate with distances between 3.80 Å to 4.80 Å (Figure 2(b)). They have no apparent obstacle to approach aminoacrylate and are most probably those that participate in hydrolysis of detached aminoacrylate to pyruvate and ammonia in the final step of the enzymatic reaction. In addition there are two, apparently important, buried localized water molecules (Figure 3), that each form multiple polar interactions with the surrounding protein residues. One of these water molecules is a hydrogen bond donor to main chain oxygen atoms of Gly64 and Ser250, and to Nε of catalytic Lys251. It serves as hydrogen bond acceptor from Oγ of Ser256 and from the main chain nitrogen atom of Gly66. The second water is a hydrogen bond donor to the main chain oxygen atoms of Met90 and Gly254 and one of the carboxyl oxygen atoms of Asp65. It serves as a hydrogen bond acceptor from the main chain nitrogen of Gly66 and Oγ of Ser256. Both these buried water molecules are located in a narrow tunnel 8 Å in length that leads from the active site to the dimer interface and are in line with the Lys251 N ε . Catalytic Lys251 makes hydrogen bond interaction with Cβ H2 of aminoacrylate, thereby putting the cofactor in “communication” with a pool of solvents. These water molecules may serve as the path

Two Structures of Alliinase from Alliium sativum L.

of a proton translocation away from the active site into a relatively large pool of water molecules that form first and second shell contact with the protein. The comparison of active site with PLP-AA and the alliinase site with internal aldimine PLP-Lys adduct In the previously reported orthorhombic alliinase dimer26 there are two independent molecules per asymmetric unit with different active site contents. The active site contents of monomer B are comprised of a mixture of several chemical species including PLP in the catalytic pocket, and each separate species was de-convoluted out of the overlapped electron density, whereas monomer A clearly shows the expected aldimine between Lys251 and PLP. A comparison of monomer A active site with its internal aldimine and the active site of the current PLPamino-acrylate complex structure has been made. For this comparison the alignment of the two active sites was done using the superposition of PLP pyridine ring, O3′, CH3 and phosphate tails (r.m.s. on 14 pairs of superimposed atoms is 0.156 Å). It is immediately obvious that no major rearrangement of the active site has taken place; however, small, but important displacements are observed in both cofactor and side-chain conformations. In terms of the cofactor, the largest difference between comparable atoms seen in the PLP pyridine ring is at the C4 position (0.3 Å). Due to a deviation from planarity at this position the C4 atoms move in opposite directions (relative to the ring plane). This deviation propagates to a difference of 0.74 Å at the C4′ atom positions (Figure 4) and puts an approximate 16° angle between C4-C4′ bonds of the two structures. The tilt of the cofactor in PLP-AA form is away from Lys251. Despite the cofactor tilt, Asp225 and Tyr228 side-chains maintain their hydrogen bonding interactions with the PLP pyridine ring N and phenolic O3. Cofactor tilt angles of a similar magnitude have been observed in other covalent active site complexes of PLP-dependent enzymes, regardless of their family.28,35–37 This tilt angle moves the aminoacrylate moiety towards the guanidinium group of Arg401 that anchors the carboxylate group

Figure 3. Stereoview of the hydrogen bonding interactions within the water channel leading from PLP-AA through Lys251 to the dimer interface.

Two Structures of Alliinase from Alliium sativum L.

617

Figure 4. Superposition of the alliinase active site residues. The PLP-AA external aldimine form is in gray and the internal aldimine form (monomer A of 1lk9) is in green. The cofactor ring tilts away from the catalytic Lys251 by approximately 16° and the lysine Nε rotates away from C4′. The sidechains of residues Asn207 and Arg401 make alternative interactions, with either the aminoacrylate moiety (external aldimine form) or with a solvent water and Hepes (internal aldimine form).

of the aminoacrylate by an extensive hydrogen bonds network and away from Lys251. In the case of the internal aldimine form of alliinase, the position, orientation and conformation of Arg401 is largely unchanged relatively to aminoacrylate form. The guanidinium group of Arg401 of internal aldimine structure is locked in the space by interactions with the side-chain oxygen atom of Asn207, and main chain oxygen atom of Ala62 and instead of carboxyl group of aminoacrylate it interacts with Hepes and a water molecule. In terms of Lys251 residues, the Cα positions in the two structures are very close (0.2 Å) and torsion angles X1, X2, X3 in both molecules are nearly identical, but the side-chains gradually diverge bringing Nε atoms 2.1 Å apart and pointing in the opposite directions (Figure 4). Active site comparison with other structures containing aminoacrylate Search of the Protein Data Bank38 revealed only two other known structures with true amino acrylate intermediates: cystine C-S lyase from Synechocystis (1ELU) and the tryptophan synthase (1A5S).39 The cystine C-S lyase from Synechocystis has a 20.3% sequence identity (32.8% similarity) with alliinase and its crystal structure has been solved with aminoacrylate in the active site.27 Despite low sequence identity the active sites of the alliinase and C-S lyase are similar enough (probably due to the similar enzymatic reaction) that comparisons can be conveniently made. In the case of tryptophan synthase39 a comparison is not discussed as the sequence and structural conservation of the active sites of these enzymes are too diverse. The alignment of the C-S lyase and alliianse active sites was made according to the procedure described in the preceding section. Superposition shows that despite different torsion angles in the phosphate tail, phosphor atoms are 0.52 Å apart, and the oxygen atoms are separated by similar shift

and an additional rotation of approximately 60°. The phosphate oxygen atoms, in both cases, are strongly anchored by multiple interactions with the surrounding protein residues (Figure 5). The catalytic lysine residues, Lys251 and Lys223, (alliinase and C-S lyase, respectively) have different torsion angles; the Cα positions are separated by 2.3 Å but the Nε positions are 0.6 Å apart. In both cases the Nε is too far from the C4′ to make covalent contact, 2.97 Å in C-S lyase and 3.41 Å in the present structure. In both active sites the guanidinium groups of Arg401 and Arg369 (alliinase and C-S lyase, respectively) make strong hydrogen bond networks to the aminoacrylate moiety carboxylate group, thereby locking it into position in a strained conformation (Figure 5). The PLP pyridine ring in the C-S lyase active site is held in position by being sandwiched between Ala199 and His114 and between residues Val227 and Tyr165 in alliinase. The Cβ of Ala199 and the Cγ2 of Val227 are positioned 0.9 Å apart and are 3.6 and 3.8 Å, respectively from the plane of the pyridine ring. Planes of His114 and Tyr165 are positioned to make stacking interaction on the opposite side of the pyridine ring and are rotated about 30° from each other. Highly conserved amongst PLP-dependent enzymes Asp225 and Asp197 (alliinase and C-S lyase, respectively) are relatively closely placed (distances between Cα atoms of 0.7 Å) and make short interactions (2.6 Å and 2.4 Å, respectively) with N1 atom of the pyridine ring. Comparison between holo and apo alliinase active site Comparison of the apo and holo structures reveals only minor rearrangments in the positions of the residues lining the active site. Despite the yellow color of the apo alliinase crystals, the active site of the

618

Two Structures of Alliinase from Alliium sativum L.

Figure 5. Superposition of the alliinase active site residues. The PLP-AA external aldimine form is in gray and the active site of PLPAA C-S lyase (1ELU) is in cyan. In both cases the cofactor ring tilts away from the catalytic lysine residue. The guanadinium groups of residues Arg401 and Arg369 make equivalent hydrogen bonding interactions with either the aminoacrylate moiety of the PLP-AA.

apo form does not contain PLP. Instead PLP is replaced by two sulfate ions, acetate and water molecules (Figure 6(a)). Superposition of the apo and holo forms was done on the basis of alignment of PLP with the sulfates and acetate (Figure 6(a)). One sulfate ion superposes exactly with the phosphate group of the PLP and maintains the same hydrogen bond network observed in the holo form structure. Two oxygen atoms of the second sulfate emulate the carboxylate group of aminoacrylate, whereas the pyridine ring of the PLP is partially mimicked by acetate and two water molecules (Figure 6(b) and (c)). Despite being small and mobile, the solute molecules in the active site of the apo structure are involved in an intricate network of hydrogen bonds and ionic interactions producing a rigid structure that mimics aminoacrylate-PLP. It was previously shown that PLP-dependent enzymes can be inhibited by sulfate ions and phosphate ions.40–43 In these studies two possible modes of inhibition were postulated: binding in the phosphate pocket or interaction with the arginine residue that binds carboxylate of the substrate amino acid. The contents of the active side in the present apo structure provide structural evidence that both modes of inhibition take place simultaneously. Comparison of the apo and holo structures reveals that the formation and binding of the aminoacrylate-PLP substrate intermediate does not induce a conformational change in the enzyme. Indeed, the alliinase active site appears to be predisposed to bind PLP and cofactor. Moreover, there is no shift in the relative positions of the large and small domains as revealed by DYNDOM.44 The location of the substrate binding domain brings Arg401 into position to bind the carboxylate group of the substrate, apparently trapped into a “closed”like form.33 However, because the apo-form active site contents replace the PLP, yielding a structure that mimics the holo structure in both form and

electrostatics, no conclusions regarding the open/ closed transition can be made. Surprisingly, the content of the apo structure active site emulates the aminoacrylate-PLP intermediate better than it emulates PLP alone. Active site and catalytic mechanism We have been able to trap the alliinase enzyme in an intermediate stage of the catalytic reaction pathway probably as a result of the crystallization in acidic conditions (pH 5.6). The reaction of alliinase on its natural substrate alliin or on (+)Sallyl-L-cysteine, which differs from alliin in that the sulfur is not a sulfoxide, is a β-elimination reaction. The structural comparison of the active sites of the internal aldimine from the previously reported alliinase structure and external aldimine PLP-AA complex structures has been made (see above). The two major structural differences between the active sites at these two stages of the enzymatic reaction are in the tilt of the PLP cofactor by 16° and in the torsion angles of the catalytic Lys251. The result of the tilt is the displacement at position C4′ away from Lys251, combined with differences in the Lys251 conformation in the current structure that generates a distance of 3.4 Å between C4′ of PLP and Nε of Lys251. In addition a large number of specific polar interactions stabilize the aminoacrylate intermediate. This information, taken together with known analogous reactions from related enzymes, allows a speculation about the reaction mechanism. In its resting state the enzyme is covalently bound to the PLP cofactor via an internal aldimine Schiff base between the catalytic lysine 251 and the cofactor C4′. The initial step in the reaction is the formation of the Michaelis complex of (+)S-allyl-Lcysteine substrate with the enzyme by a transaldimation reaction. The guanadinium group of Arg401 anchors the (+)S-allyl-L-cysteine carboxylate group

619

Two Structures of Alliinase from Alliium sativum L.

and the Gly64 main chain O and Asn207 interactions allow the formation of a Michaelis complex by positioning the allyl-cysteine substrate in the proper orientation for the initial transaldimation to take place. The hydrogen bonding and salt-bridges from the substrate carboxylate group to Arg401 are optimized into a co-planar orientation with the

pyridine ring of PLP. In addition the residue Asn207 mediates between Arg401 and the phenolic oxygen of the cofactor and may relay information about pyridine ring conformation via Arg401 to the substrate. The (+)S-allyl-L-cysteine substrate amino group makes a nucleophilic attack of the C4′ of the internal aldimine, with the concomitant release of Lys251 from its Schiff base. At this stage there is now a covalent external aldimine linkage between the substrate and the PLP. The steric constraints of the active site pocket stabilize the Cβ–Sγ bond of the bound substrate perpendicular to the plane of the PLP. Subsequently Cβ–Sγ is lysed, with the Lys251 assumed to be acting now as the catalytic base. 45 As in other PLPdependent enzyme systems, the charge on the PLP cofactor is dissipated due to N1 charge stabilization by the totally conserved residue Asp225, and pistacking interactions between delocalized pi-system of the cofactor and Tyr165. This C-S lysis releases allyl mercaptan, however, no elimination product could be identified in the crystal structure. The catalytic Lys251 ε-amino group abstracts a proton from Cα to form the aminoacrylate-PLP intermediate captured in our crystal structure. In the acidic crystallization conditions (pH 5.6) it may be that reverse transaldimation becomes rate limiting. Moreover, in the present holo structure the tilt of the cofactor introduces too great a distance between the Lys251 Nε and the C4′ and even a series of Lys251 side-chain torsion angle rotations would not be sufficient to bridge this gap. The final nucleophilic attack of Lys251 NH2 on C4′, or reverse transaldimation, to restore the Schiff base, does not takes place and the iminopropionate is not released from the cofactor. The substrate that underwent molecular reaction to form aminoacrylate in the present holo structure may have come from one of two possible sources. It may have been the (+) S-allyl cysteine used as a crystallization additive; however, we cannot rule out the possibility, that the naturally occurring alliin from the garlic bulbs that in contact with the enzyme during the protein production and purification is the source of the observed PLP-AA intermediate. Glycosylation Four potential N-linked glycolsylation sites are identified per monomer of alliinase: Asn19, Asn146, Figure 6. (a) Superposition of the alliinase active site residues. The PLP-AA external aldimine form is in gray and apo form (monomer A of 1LK9) is in green. In the apo form the positions of the phosphate group of the PLP and the aminoacrylate moiety (external aldimine form) are replaced by SO4, while maintaining interactions with Arg401 and the phosphate binding residues (not shown). Acetate and solvent water molecules partially replace the pyridine ring of the PLP. (b) Likelihood weighted 2Fo–Fc electron density in the region of the monomer A active site of the PLP-AA form contoured at 1.0 σ. (c) Likelihood weighted 2Fo–Fc (σA weighted??) electron density in the region of the monomer A active site of the alliinase apo form contoured at 1.0 σ.

620 Asn191 and Asn328.8,46,47 Electron density is clearly visible for carbohydrate atoms attached to two out of these four sites, i.e. Asn146 and Asn328, allowing identification of composition and connectivity of the sugar moieties. A varying degree of glycosylation is seen at these two positions, dependent upon the monomer and crystal form. In the vicinity of Asn191, additional weaker electron density is seen, that can be interpreted as carbohydrate moiety attached in monomers A and B of the holo-form only. No carbohydrate density is seen at Asn19 in any of the four independent monomers of the holo-form or in the apo structure.

Two Structures of Alliinase from Alliium sativum L.

The quality of the electron density for carbohydrates in monomer A is clear, revealing a branched hexasaccharide N-linked to Asn146 (Figure 7(a) and (b)) in both holo and apo-forms. It adopts a fairly extended conformation with the first three sugars of the main chain nearly co-planar and the terminal sugar nearly perpendicular to them. The two branched sugars are positioned one above and the other below this plane. The first sugar is the N-linked GlcNAc linked by 1,4β to GlcNAc and a 1,3α linkage to a fucose. The second GlcNAc is linked 1,4β to mannose. The mannose connects 2-1β to xylose and 3-

Figure 7. (a) Word structure of the oligosaccharide found at the glycosylation site Asn146 of monomer A showing the connectivity and identification of the individual sugar moieties. For comparison the structure of 000.1FX is shown. (b) Stereo view of the | 2Fo–Fc| electron density map contoured at 1.0 σ in the region of the glycan junction between monomers A and C. The Cα trace of monomers A and C are given in green and blue, respectively. Shown in red is the final model of the hexa-saccharide chain that is attached to Asn146 of monomer A, and bridges to Monomer C. Prepared with PyMOL.70

621

Two Structures of Alliinase from Alliium sativum L.

1α to the terminal mannose (Figure 7(a) and (b)). The only difference between this oligosaccharide and the “short-chain complex, vacuole type” 000.1FX, oligosaccharide most commonly seen in plant glycoproteins48 is the lack of one additional mannose with a 61α linkage (Figure 7(a)). In reality, the electron density map in this region may suggest additional atoms, which could be interpreted as either a severely disordered additional sugar moiety, or an extended chemical modification on the sugar ring; however as it could not be positively identified, it was not modeled. The glycosylation sites on alliinase are found on the surface of the protein pointing into bulk solvent region, and as such, the majority of hydrogen bonds with the sugars are to solvent molecules. However, the oligosaccharide attached to Asn146A traverses the intermolecular space between the two dimers AB and CD, respectively, of the asymmetric unit (Figure 7(b)), and is anchored by hydrogen-bond interactions to both monomers A and C. These hydrogen bonds have the effect of tethering the end of the oligosaccharide chain to the protein, perhaps stabilizing the conformation, which makes the electron density so clear. In particular, the xylose Xys504 O3 and O4 interact directly with Lys266C main chain O and Asp123C main chain O, respectively. Mannose Man505 makes direct H bond between O3 and NZ of Lys266C and Man505 O4 binds to OD1 of Asp241C. Due to the spatial arrangement of the two crystallographically independent dimers AB and CD in the monoclinic holo-form, the distance between monomers A and C is shorter than the distance between the monomers B and D. The sugar chain attached to Asn146A is in close contact with monomer C, whereas the equivalent chain attached to Asn146B, is not within bonding distance to monomer D. As such only two sugar moieties (GlcNAc and Fuc) are seen linked to Asn146B. Similar situation is seen for the glycosylation sites Asn146C and Asn146D in the dimer CD, for Asn146C four sugar moieties are seen (GlcNAc, Fuc, GlcNAc and Man) and for Asn146D three sugar moieties are seen (GlcNAc, Fuc, GlcNAc). Glycosylation site Asn328 shows on all four monomers shorter oligosaccharide chains: three for Asn328A (GlcNAc, GlcNAc and Man) and for the Asn328B, C and D disaccharide GlcNAc and GlcNAc. The asymmetry in the degree of observed glycolsylation may be an effect of the crystal packing environment. The relatively low crystallographic symmetry of the current structure (space group P21) combined with the position of the four independent molecules of the asymmetric unit, means that the chemically equivalent sugar chains of the dimer are actually in crystallographically different environments. In the previously reported orthorhombic alliinase structure26 (1LK9) a similar pattern of glycosylation was observed. At Asn146 both structures are most heavily glycolsylated (six sugar moieties in the present structure and four in the earlier reported structure), and comparison shows that the saccharide chains diverge by approximately 14°.

The functional role of the carbohydrate moieties may be for the binding of alliinase to lectins (ASAI and ASAII)47,49–51 that co-exist in garlic bulb cells. ASAI was shown to have multiple oligosaccharide binding sites, a total of six per lectin dimer.52,53 These lectins have been shown to have very high specificity for high mannose oligosaccharide chains,54 including chains of the type as identified bound to Asn146 in this alliinase structure. However additional functions for the oligosaccharide, such as the trafficking of the alliinase to the site of the intracellular localization, should not be ruled out, as there is no known physiological functional role of the alliinase-ASA complex.55 Conclusions The high resolution structure of an apo-form of alliinase from Alliium sativum L. was determined and compared with the high resolution structure of alliinase complexed with an aminoacrylate-PLP reaction intermediate. The composition of the active site contents in both structures was unambiguously determined. The apo form provides direct structural observation of inhibition by sulfate ions. In addition, the external aldimine of the complexed alliinase structure was compared with a previously reported alliinase structure containing an internal aldimine. Both the apo and holo forms of alliinase presented here are glycoslyated, providing a unique opportunity to observe the geometry and conformation of extended N-linked hexa-saccharide chains.

Materials and Methods Preparation of holo-enzyme alliinase Alliinase was purified from garlic cloves following previously described protocol8 with an additional final step. Briefly, peeled garlic cloves were homogenized in the cold in a mincing machine in 0.02 M sodium phosphate buffer (pH 7.2), containing 10% (v/v) glycerol and 0.02 mM pyridoxal 5′-phosphate (buffer A). The homogenate was filtered through two layers of cheesecloth and the filtrate was centrifuged at 20,000g for 30 min at 4 °C. Polyethylene glycol (PEG) 8000 was added to the supernatant (to 25% w/v) and the mixture was stirred slowly for 20 min at 4 °C. The slurry was then sedimented at 20,000g for 15 min at 4 °C. The pellet was resuspended in 120 ml of 0.02 M Hepes buffer (pH 7.2) containing 1 mM CaCl2, 1 mM MnCl2, and 0.5 M NaCl (buffer B), subjected to centrifugation again at 20,000g for 20 min at 4 °C and dialyzed against the same buffer. The supernatant was placed on a ConA-Sepharose (Pharmacia) column (2.2 cm × 50 cm) pre-equilibrated with buffer B. The column was washed with buffer B. Elution of alliinase was carried out with 0.1 M methyl α-D-mannoside in buffer B. Solid (NH4)2SO4 was added to the eluate to a final concentration of 1 M. The pH of the solution was adjusted to 6.5. This preparation was placed on OctylSepharose column (1.0 cm × 10 cm) and fractions containing non-adsorbed protein were collected, dialyzed against 0.02 M Hepes buffer (pH 7.2), concentrated and stabilized by the presence of 20% (w/v) sucrose. Alliinase activity

622

Two Structures of Alliinase from Alliium sativum L.

was assayed according p-nitrothio benzoate (NTB) method.56 Protein was assayed by the Lowry procedure57 with ovalbumin as a standard. Specific activity of the alliinase preparation obtained was 250 μmol min−1 of pyruvate per 1 mg protein. These tests were done to ensure that that the enzyme to be used for crystallization was fully functional.

line ID29 at ESRF (λ = 0.976 Å). A low resolution data set was collected from each holo and apo enzyme crystal using larger distance, attenuated beam and shorter exposure time. The diffraction data were processed and reduced with DENZO and SCALEPACK incorporated into HKL2000 package.58 The crystal and data parameters are summarized in Table 1.

Preparation of apo-enzyme alliinase

Structure solution

The apo-enzyme alliinase was prepared by removing the PLP cofactor from the holo-enzyme using a procedure in which holo-enzyme was treated with hydroxylamine. The holo-enzyme in PBS buffer with 25% (v/v) glycerol was mixed with 100 mM hydroxylamine and 20% (w/v) ammonium sulfate and incubated at 37° for 3 h. The reaction mixture was then applied to a Sephadex G-25 column. Protein was monitored by measuring absorbance at 280 nm. Evidence for the removal of coenzyme PLP in the apo-enzyme form is provided by the UV spectral analysis. The apo-alliinase prepared did not show any significant absorption at 430 nm and not more than 5% of enzyme activity.

The monoclinic holo alliinase crystal structure has four independent molecules in the asymmetric unit. The structure was determined by molecular replacement (MOLREP)59 using a monomer of the published26 structure of an orthorhombic form of garlic alliinase (PDB code 1LK9) as a search model, using resolution range of 46.0 to 4.0 Å. The cross-rotation function revealed four peaks of approximately 10 σ with the next peak in the list of 4 σ. Translation search for four independent molecules gave a clear solution with an R factor of 0.36 and correlation coefficient = 0.62. The tetragonal apo-form alliinase structure was solved by molecular replacement (MOLREP)59 using the monoclinic holo-form monomer A, as a search model, using resolution range 40.0–4.0 Å. The cross-rotation function revealed a peak of 14 σ with the next peak in the list of 4 σ. Translation search gave a clear solution with an R factor of 0.35 and correlation coefficient = 0.65.

Crystallization of holo alliinase The monoclinic crystals were grown from 30% PEG 4000, 200 mM ammonium acetate, 100 mM tri-sodium citrate (pH 5.6), 20 mM (+) S-allyl-L-cysteine. Crystals took seven to ten days to appear and grew as dendrites. After several cycles of seeding, crystals gained considerable morphological enhancement yielding perfect prisms of yellow-golden color induced by PLP. However, in the end, the crystals that diffracted best in terms of resolution, spot shape and mosaicity were the sharp point pieces of the dendrites. Crystallization of apo alliinase Tetragonal crystals were grown from Hampton Crystal screen condition number 39, 0.1 M Hepes (pH 7.5), 2% (v/v) PEG 400 and 2.0 M ammonium sulfate. The crystals took between four to six months to appear and grew to a maximum size of 0.1 mm × 0.1 mm × 0.1 mm. Data collection Holo and apo enzyme crystals were delivered to the synchrothron (ESRF) packed with fresh mother liquor in sealed glass capillaries. When the capillaries containing holo enzyme crystals were opened for use, a strong distinctive odor of garlic was released, indicative of alliinase enzymatic activity, despite the fact that this odor was not sensed during crystal packing. Capillaries containing apo-enzyme crystals were odorless. Prior to data collection under cryogenic conditions, both crystals were transferred to Hampton paratone-N oil for a period of 10 min before being mounted in a rayon fiber loop and flash cooled in the flow of cold nitrogen at 100 K. A full data set from one holo enzyme crystal to 1.39 Å resolution was collected as 0.5° oscillation frames, 5 s/ frame, at a distance of 120 mm with an ADSC CCD detector at beam line ID14-2 at ESRF (λ = 0.933 Å). A full data set from one apo enzyme crystal to 1.60 Å resolution was collected as 0.5° resolution frames, 1 s/frame at a distance of 140 mm with an ADSC CCD detector at beam

Structure refinement The same refinement/model-re-interpretation protocol was used for both structures. After molecular replacement 20 cycles of the rigid body and ten cycles of the restrained refinement were executed using REFMAC560 as implemented in CCP4,61 with CCP4i.62 A random set consisting of 5% of the reflections was excluded from calculations for crossvalidation of various refinement strategies63 such as geometric and temperature factor restrains (re-assessed on various stages of refinement), sugar and solute molecules insertion and basis for maximum-likelihood refinement protocol implemented in REFMAC5.60 To estimate the potential limit of the model convergence and to eliminate model bias, ARP/WARP 6.0 software suite for improvement and objective interpretation of crystallographic electron density maps and automatic construction and refinement of macromolecular models, was implemented using mode of maps improvement by atoms update and refinement.64,65 The resulting coordinate file was a hybrid in a sense that in addition to atoms of the protein molecule it contained dummy atoms that modelled features of the electron density map dissimilar to the model or missing from the model: water and other solute molecules and alternative conformations. This hybrid coordinate file was input to ten cycles of REFMAC5 refinement. At this stage the refinement protocol resulted in R factor = 17.4% (Rfree = 20.1%) and R factor = 17.3% (Rfree = 22.1%) for holo and apo alliinase, respectively. The electron density maps were calculated with likelihood weighted 2|Fo|–|Fc| and |Fo|–|Fc| coefficients and the protein structure was re-interpreted using COOT.66 The PLP aminoacrylate derivative was unambiguously detected in the active site of the holo alliinase. In the active site of apo alliinase two SO4 and one acetate groups were detected together with several water molecules. Sugar moieties were located and identified by inspection of likelihood weighted 2|Fo|–|Fc| and |Fo|–|Fcalc| electron density maps. The dummy atoms were deleted from the coordinate list and the improved model gradually

623

Two Structures of Alliinase from Alliium sativum L. emerged during cycles of rebuilding/refinement. Clear electron density representing alternative conformations was observed near several amino acid chains, which were subsequently modelled as alternative, mutually exclusive conformations. Water molecules were assigned initially automatically using COOT.66 At the later stage of water insertion, difference electron density map peaks were inspected manually and the decision to insert them was based on the electron density peak's appearance, value, location and distance from the nearby structural elements. Idealised hydrogen atoms were inserted and refined in the riding mode. In the holo alliinase structure thermal parameters were refined anisotropically and in apo alliinase isotropically. The occupancy factors of solvent molecules were assumed to be unity, except for water molecules associated with the alternative sidechain conformations, where they were set according to alternative side-chain occupancy. Scaling parameters, bulk solvent correction, atomic coordinates, TLS parameters,67 anisotropic temperature parameters for holo alliinase and isotropic thermal parameters for apo alliinase were refined during the last cycles of refinement. The final cycles yielded R = 16.9% (Rfree = 20.5%) and R = 16.4% (Rfree = 17.9%) for holo and apo structures, respectively. Quality assessment was done using PROCHECK,68 WHATIF69 and COOT.66 Refinement parameters are summarized in Table 1. Protein Data Bank accession codes Atomic coordinates have been deposited with the RCSB Protein Data Bank under accession codes 2HOX and 2HOR for holo and apo structures, respectively.

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Acknowledgements The authors are grateful to the European Synchrotron Radiation Facility (ESRF, Grenoble, France) for synchrotron beam time and staff scientists of the ID14 stations cluster for their assistance. This publication is dedicated to the memory of the late Elizabeth V. Goryachenkova.

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Two Structures of Alliinase from Alliium sativum L.

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Edited by R. Huber (Received 25 September 2006; received in revised form 5 November 2006; accepted 9 November 2006) Available online 14 November 2006