Proc. Natl. Acad. Sci. USAVol. 77, No. 5, pp. 2559-2563, May 1980Biochemistry

Three-dimensional structure of a pyridoxal-phosphate-dependentenzyme, mitochondrial aspartate aminotransferase

(protein structure/active site/x-ray diffraction)

GEOFFREY C. FORD, GREGOR EICHELE, AND JOHAN N. JANSONIUSAbteilung Strukturbiologie, Biozentrum der Universitit Basel, CH-4056 Basel, Switzerland

Communicated by Esmond E. Snell, February 14,1980

ABSTRACT X-ray diffraction studies to 2.8A resolutionhave yielded the three-dimensional structure of mitochondrialaspartate aminotransferase (L-aspartate:2-oxoglutarate ami-notransferase, EC 2.6.1.1), an isologous a2 dimer (M, = 2 X45,000) The subunits are rich in secondary structure and containtwo domains, one of which anchors the coenzyme, pyridoxal5'-phosphate. Each active site lies between the subunits and iscomposed of residues from both of them.

Aspartate aminotransferase (AATase; L-aspartate:2-oxoglu-tarate aminotransferase, EC 2.6.1.1) is the most studied of thevitamin B-6-dependent enzymes (1, 2). These enzymes catalyzea wide variety of transformations in amino acid metabolism.AATase effects the reversible transfer of an amino group fromL-aspartate or L-glutamate to the a-keto acids a-ketoglutarateand oxalacetate. In the course of the double displacement re-action (3, 4) the coenzyme shuttles between the pyridoxal-Pform (bound via an aldimine linkage to the E-amino group ofa lysine) and the pyridoxamine-P form. "Syncatalytic" con-formational changes occur in the enzyme matrix (5-7). Variouscoenzyme-substrate intermediates are identified by charac-teristic absorption and circular dichroism spectra (2, 8). Thestereochemistry of pyridoxyl-catalyzed reactions has beenprobed (9), and a dynamic reaction mecha'nism has been pro-posed (10).Two homologous, genetically independent isozymes of

AATase have been found in animal tissues, one in the cytosol(cAATase), the other in the mitochondrial matrix (mAATase).Both are a2 dimers of about 2 X 400 amino acids. The aminoacid sequences of the pig (11-13) and chicken (refs. 14 and 15;U. Hausner, K. J. Wilson, and P. Christen, personal commu-nication) isozymes are known or have been almost completelyelucidated. Crystallized AATase offers an exceptional oppor-tunity for the study of interactions amongst protein, coenzyme,and substrates during catalysis. The crystalline enzyme is cat-alytically competent (16). Single-crystal microspectrophoto-metric studies on cAATase (17, 18) and mAATase (16, 19) havepermitted the recognition of several reaction intermediates andthe evaluation of some dissociation constants and kinetic pa-rameters (19). Now a detailed picture of the enzyme isemerging from X-ray studies of such crystals. The high-reso-lution X-ray analyses of cAATase from chicken (20) and pig (21)are near completion. Here we report the three-dimensionalstructure of chicken mAATase in the internal aldimine form(pyridoxal-5'-P-enzyme) as revealed by a 2.8-A resolution X-raystudy.

RESULTSStructure determinationChicken mAATase crystallizes in a triclinic unit cell that con-tains one a2 dimer (22) whose subunits are related by a non-crystallographic dyad (23). Diffraction data to 2.8-A resolutionof the native protein and the derivatives listed in Table 1 werecollected on a CAD4F diffractometer (Enraf-Nonius, Delft,Netherlands). Previously found heavy atom sites (23) were re-evaluated by difference Fourier maps. They were refined byalternate cycles of multiple isomorphous phase evaluation (24)and least squares refinement (25) with a separate occupancyfor each site in each crystal (26). The overall figure of merit inthe 2.8-A study was 0.80, and it was 0.75 in a 3.2-A study withfour derivatives in which only site occupancies had been re-fined.An electron density map of the native structure was calcu-

lated by using centroid phases (24) and, as in our earlier work,was displayed in an orthogonal grid (X',Y',Z'). The Z' axis is themolecular dyad relating the two subunits, S and S*, while theX' axis runs along the length of the dimer (23). The 3.2-A res-olution map could be interpreted readily as the folding of twoisologous polypeptide chains that had strong density along theirbackbones and unusually well-defined side chains. The phos-phate and pyridoxyl moieties of the coenzyme were clearlyvisible and the Schiff base linkage to Lys-258 was unambigu-ously defined. The knowledge of the sequences of pig andchicken mAATases, which are about 85% identical, was in-valuable. The residue names of pig mAATase of ref. 12 areused, renumbered as in Fig. 1. These 401 amino acids and theside chain densities encountered along the polypeptide chaincorrespond. No density was found for the two additional resi-dues, 131 and 132, of the alternative pig mAATase sequence(13). A backbone model of the protein was fitted (27) to esti-mated a-carbon positions in the 3.2-A resolution map. Anatomic model of the active site of subunit S was built fromKendrew-Watson parts, using an optical comparator (28) withthe mirror parallel (29) to sections of the 2.8-A resolutionelectron density.Description of the structureA subunit of mAATase is compact and its envelope resemblesa cashew nut (Anacardium occidentale) with dimensions 70X 50 X 40 A. The subunits overlap around the molecular dyadand produce a dimer, 105 X 60 X 50 A. The top and sides of themolecule (see Fig. 2) are composed of helices, whereas thebottom is formed by strands of ,B sheet and extended hairpin

Abbreviations: AATase, aspartate aminotransferase; mAATase, mi-tochondrial AATase; cAATase, cytosolic AATase. The atoms in pyri-doxal-5'-P are numbered according to the IUPAC-IUB rules (32).

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Table 1. Heavy atom derivatives and refinement statisticsResolution, Param- Derivative

A eter* CMMPUt Ptent Na2IrCl6t KReO4t MeHgI Pt(NH3)2C1214.0-4.5 (tYH) 1606 1551 1123 914 1783 1794

(E) 489 659 615 437 759 787

4.5-3.2 (JH) 1294 1164 706 958 1022 744(e) 653 794 612 576 697 696

3.2-2.8 (fH) 795 622 742 721 529(,e) 690 562 483 718 650

No. ofsites 11 18 18 1 12 16

CMMPU, 3-chloromercury(II)-2-methoxypropyl urea; Pten, platinum ethylenediamine dichloride;MeHgI, methylmercury(II) iodide.* (fH) is the root mean square heavy atom structure factor amplitude and (e) is the root mean square"lack-of-closure" in arbitrary units. The mean structure factor amplitudes of the native protein inthe same resolution ranges are 6361/5836/4205 for 4739/8157/5596 reflections. The correspondingmean figure of merit is,0.89/0.79/0.72 with an overall value of 0.80.

t Used in 3.2-A resolution study.

loops. The subunit can be dissected, somewhat arbitrarily, intofour parts: a large domain, which binds pyridoxal-P and con-tains residues 76-300; a small domain, which is composed ofresidues 15-47 and 359-410 (the COOH terminus); an NH2-terminal arm of residues 3-14; and, finally, a bridge across thetop of the two domains, which is formed by residues 48-75 and301-358.The pyridoxal-P-binding domain (Figs. 2 and 3) includes a

seven-stranded a/13 fold in which the chain passes alternatelythrough helices and strands of sheet. Its topology is novel [+5x,+lx, -2x, -lx, -lx, -1 in Richardson's (30) notation]. Allcross-overs are right-handed and are made by helices. Thestrands (a, g, f, e, d, b, c) of the :3 sheet have directions (+, -,+, +, +, +, +). Their twist is in the usual sense (31) and pro-duces a left-handed rotation of some 1000 from one end of thesheet to the other. Two hairpin loops are found, around residues163 and 196. One runs beside the small domain, the otherprojects into the active site. In the pyridoxal-P-enzyme, thecoenzyme lies against strands e and f of the : sheet.

Four extended strands, enclosing a hydrophobic core, formthe heart of the small domain. The strands associate in pairs,(A, C) and (D, B), with directions (+, +) and (-, +), respec-tively. Their mutual interactions are complex and must bedetermined by model building. The domain combines twoseparate segments of the polypeptide chain. Residues 15-35 liedown strand A and an associated helix a(22) (in the notationof Fig. 1) whereas strands B, C, and D and two additional helicesare part of the sequence 359-410. The NH2-terminal arm,

residues 3-14, extends from the small domain of subunit S, infront of the active site of S, and terminates against the undersideof the pyridoxal-P-binding domain of S*.

Within one subunit, the domains are bridged twice. Onechain segment, residues 47-75, passes beside the active siteregions of both subunits. It runs across the top of the active siteof S, through helix a(58), and dips into the active site of S*. Asecond segment, residues 301-356, folds into three contiguoushelices. One of these, a(329), is some 50 A long. Helix a(356)and part of a(329) lie on the exterior surface of the pyridoxal-P-domain, whereas the COOH-terminal half of a(329), helixa(353), and the hairpin loop around residue 163 form a wallthat borders the small domain.Active siteThe approaches to the active sites of mAATase are the deep riftsthat border the subunit-subunit interface. Each active site iscomposed of structural elements of both subunits. We describethe active site of subunit S in the orientation of Fig. 3. Itsboundaries are: on the left, residues 276*-301* which builda(284*) and a chain running beside the Z' axis; on the right,residues 380-390 from the small domain of S; in front, theNH2-terminal arm; below, helix a(146); above, residues 30-60,which wind across the top of the small domain and run intoa(58). The coenzyme is anchored to the pyridoxal-P-bindingdomain of S at the back of the active site cleft and lies flatagainst the COOH termini of strands e and f. The 3 sheet curlsin such a way that strands b and c run far beneath the active site.

1 2 3 4 5 6 7 8 9 0 1 2 3 4 5. . . . . . . . . . . . .

t t t t . ... H H H HH H H H H H H H H . . H H H HH H H H B BEE B BEI B B BL L L L L L L .H H H H H H H. H H H H H H H H H H H H H HH H H H H H . . BBB B B BBH H H H H H H H H H H HH H H H H H H H H H H H H H HE E E E . . H H H H H H H H HH H H H H H E H H H I I

6 7 8 9 0H H H H HH H H H HH H H B Bt t t * .

H H HH .

H 8 BBBBh h hh h

H H H H H. . E EE

1 2 3 4 5 6 7 8 9 0 1 2 3 4H H H H H H H H . . EE EEH H H . E ..... . t t tB B B 8 B . .H H H H H H H.H H H H H H H H H .B B. BB B BB . . . . L L LB 8 BB . . . . . . . H H H...... ......BB B B B BB

H H H H H H H H H H H . H HHHH . .... H H H H H H HE E t t t E E E E E . . . H

5 6 7 8 9 0E ....t . HHHHHHHHHHB B BB .1LLLLL.HHHHHH0HHHHHHHHHHHHHH t tEHHHHHH

FIG. 1. Assignment of secondary structure. The residue numbers are those of the pig cAATase sequence (11). The gaps (I) in the pig mAATasesequence (12) are inserted to maximize the degree of sequence similarity to cAATase. The symbols indicate the conformation assigned to theresidues: H, helical; B, sheet in pyridoxal-P-binding domain; E, extended chain in small domain; L, hairpin loop; t, tight turn; h, one-turnhelix; ., no structure assigned. In the text, a helix with residue i at its midpoint is designated helix a(i).

04080120160200240280320360400

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FIG. 2. Stereo drawing of thebackbone of the mAATase dimer.Subunit S (double lines), its isolo-gous counterpart S* (single lines),and the coenzyme with its Schiffbase C=N bond (solid bonds) areshown. The essential lysine ismarked (-). Large circles corre-spond to numbered residues. Theorigin (9) of the orthogonal mo-lecular axes is indicated and the Z'axis points towards the reader. Inour convention, this view of themolecule is "from above."

The NH2 terminus of a(116) makes contact with the coen-

zyme's phosphate group. Residues 255-260 form a one-turnhelix beneath helix a(58). Residues 68*-72* dip into the activesite in front of the coenzyme.

In the active site model of the pyridoxal-P-enzyme, thephosphate group is somewhat in front of the pyridine ring. TheSchiff base and the pyridine ring are roughly coplanar and tiltsome 20° away from the X'-Z' plane (see Fig. 2). The aldiminelinkage is cisoid-i.e., directed towards the 3 hydroxyl ofpyridoxal-P. The pyridoxyl group is oriented with its A faceagainst the ,3 sheet and its B face partially exposed to solvent.[We denote as A and B faces the faces of the pyridine ring aboveand below, respectively, the plane of the paper in the standardrepresentation (32). The cisoid aldimine linkage equates thesi side of that C4'=N bond with the A face of the ring.] Theindole group of Trp-140 lies in front of the pyridoxyl moiety,tilted some 600 away from it. The tryptophan covers the lowerpart of the B face of the pyridine ring and the three imidazole

rings of His-143, His-189 and His-193 that are parallel and liebelow the pyridoxal-P. These three histidyl residues are the onlyones observed in the active site region. Asp-222 is hydrogenbonded to the protonated (33) N' of pyridoxal-P, and probablyto Nd2 of His-143. Tyr-225 approaches from behind and itshydroxyl group forms a hydrogen bond to the ionized (34) 3

hydroxyl of pyridoxal-P. The CO atom of Ala-224 lies againstthe A face of the pyridine ring and blocks its backward move-

ment. The phosphate group of the coenzyme in anchored in thestrongly polar pocket formed by the guanidinium group ofArg-266, the : hydroxyl of Ser-255, and main chain and sidechain atoms of residues 107-109. The phosphate charge isbalanced by an ion pair with Arg-266 and by interaction, in themanner proposed by Hol et al. (35) with the dipole of helixa(l16).

Several side chains project from the walls of the active siteinto the solution in front of the coenzyme. Asn-194 lies to theright of Trp-140 and its side chain is close to the 3 hydroxyl of

51

395

*A6 / 30

4 z FIG. 3. Stereo drawing of theactive site (of subunit S) of mAA-

376 Tase. The coenzyme with its Schiff

353 , base C=N bond (solid bonds) and

63x parts of the backbones of subunits5L;S (double lines) and S* (single20;384 lines, # after the numbers) are

2438

shown. Large circles correspond to1381 61 residues explicitly mentioned in

the text. The view is directly alongthe molecular Y' axis. The seven-

215 stranded sheet of the coen-

180 zyme-binding domain is clearlyvisible.

Y.

x. X.

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pyridoxal-P. The guanidinium groups of two arginines arepositioned on the same level as, and somewhat in front of, thecoenzyme. These residues are, on the right, Arg-386 from thesmall domain of S and, on the left, Arg-292* from helix a(284*)of S*. Another residue, Tyr-70*, extends from S*, and its sidechain points towards the nitrogen of the aldimine linkage. Theseexplicitly identified residues are conserved in the knownAATase sequences (11-15) and, we believe, play a prominentrole in the catalytic function of the enzyme.

DISCUSSIONProteins that have evolved from a common ancestor seem toretain a single spatial structure in which the divergent aminoacid sequences are accommodated. The mAATases and cAA-Tases are genetically independent isozymes whose interspeciessimilarities exceed those of the mitochondrial and cytosolicisozymes of one species (2). The amino acid sequences ofAATase (11-15) show an identity of 82% between cAATase-(chicken) and cAATase(pig), of about 85% between mAA-Tase(chicken) and mAATase(pig), and of 48% betweenmAATase(pig) and cAATase(pig). Recently, we have shownthat mAATase(pig) and mAATase(chicken) have identicalchain folds and, in a 3.2-A resolution difference map betweenthe two structures, we have observed strong local electrondensity differences that could be associated with substitutionsof individual amino acid side chains (36). The structures ofcAATase(chicken) and mAATase(chicken) as seen at low res-olution are closely related (20, 23). The dimers have the sameoverall shape and the helices on their surfaces form similarpatterns. The mAATase chain fold can accommodate thecAATase sequence because the additional 11 residues are in-sertions (see Fig. 1) in chain segments on the surface of themolecule. Two residues are inserted at the NH2 terminus, threeat the COOH terminus, one just after helix a(58), and fiveresidues-127, 128, 131, 132, and 153-into two adjacent 3strands, b and c, on the underside of the pyridoxal-P domain(see Fig. 3). Taken together, these observations suggest stronglythat AATase possesses a conserved tertiary structure whosemodulation, by amino acid substitutions, produces the partic-ular properties of the different enzymes.The folding pattern of the pyridoxal-P domain of AATase

is novel but may be expected to recur in other B-6-dependentenzymes, just as the dinucleotide-binding fold (37) is repeatedin NAD-dependent dehydrogenases. A conserved mode ofbinding of pyridoxal-P to apoenzyme has been proposed (38)to explain the consistent stereochemistry of B-6-catalyzed re-actions. On the other hand, we see a clear structural differencebetween AATase and the exceptional pyridoxal-P-dependentenzyme glycogen phosphorylase (EC 2.4.1.1), in which thepyridoxyl aldehyde group does not play a catalytic role (39).In phosphorylase (40, 41), pyridoxal-P is attached to the helixlinking strands D and E of a six-stranded (C, B, A, D, E, F) a/3structure that resembles the dinucleotide-binding fold (37). Thepyridoxal-P domains can be aligned by the topological equiv-alence (30) of fragment (f, e, d, b, c) in AATase and fragment(F, E, D, B, C) in phosphorylase. It then becomes clear that theassociation of pyridoxal-P and its binding domain is very dif-ferent in the two enzymes and, indeed, that the pyridoxal-P-binding sites lie on opposite sides of the 3 sheet. This funda-mental difference in structure rules out any evolutionary re-lationship between the pyridoxal-P domains of these two en-zymes.mAATase is an isologous a2 dimer. The functionally inde-

pendent (42, 43) active sites lie on opposite sides of the molecule.Each is formed by the juxtaposition of essential residues fromboth subunits and, of necessity, is incomplete in the monomer.

This situation is unusual but also occurs in glutathione reductase(44) and glucose-6-phosphate isomerase (45). It accounts for theobserved inactivity of dissociated subunits (46). The AATasestructure is compatible with the results of many studies on bothAATase isozymes: (i) The coenzyme is covalently bound toLys-258 (47) in the pyridoxal-P-enzyme and Arigoni andAustermuhle-Bertola (48) have shown that the re side of thisaldimine linkage is reduced by NaBH4. Our model agrees,because the A face of the pyridine ring and the si side of theC4'=-N bond are shielded from solvent. (ii) Identified residuesin or near the active site include: Arg-292 as a substrate-an-choring site (49); Cys-390 (5, 50) and an associated tyrosine (5),Tyr-40 (51); Tyr-70 (we have Tyr7O*) and also Cys-390, whichwere specifically crosslinked to Lys-258 by a bifunctional re-agent (52). (iii) Chemical or spectroscopic results have suggestedthat the following groups lie in the active center: a tyrosine byspecific nitration in the apoenzyme (53), a tryptophan by flu-orescence quenching upon pyridoxal-P binding (54), a histidineby photooxidation (55), a "positive pocket" around the coen-zyme phosphate (56), and an arginine (49,57,58) as the anchorof the substrate w-carboxyl group (58). Our model providescandidates for these groups: Tyr-225, Trp-140, His-143, Arg-266, and Arg-292*. In mAATase, the sequence -Ser-X-X-Lys-(-pyridoxal-P) which has been observed in several B-6-enzymes(59) forms a turn of helix with Ser-255 and Lys-258 spatiallyadjacent and associated with the coenzyme phosphate andpyridoxyl groups, respectively. The presence of all these resi-dues in the active site model of mAATase is further evidencein favor of a conserved AATase structure.A catalytic mechanism (G. Eichele, G. C. Ford, J. N. Janso-

nius, J. Kirsch, and P. Christen, unpublished work) is beingdeveloped that acts within the framework of the residues in theactive center. The mode of substrate binding in the Michaeliscomplex is evident when the active site model and differenceelectron density maps of various enzyme derivatives (unpub-lished work) are correlated. The substrate w-carboxyl groupforms an ion pair with Arg-292* while the a-carboxyl groupinteracts with Arg-386 and Asn-194. The substrate amino grouppoints towards the aldimine linkage. This arrangement orientsthe substrate Ca-H bond parallel to the Z' axis and places it nearTyr-70*. No histidine side chain is found in this vicinity.Therefore, in mAATase, a mechanism in which an imidazoleacts directly as a proton donor/acceptor (60) must be ruledout.The succeeding steps in the catalysis probably involve two

levels of enzyme rearrangement. One is a reorientation of thepyridine ring in a manner similar, but not identical, to thatproposed by Ivanov and Karpeisky (10). The ring moves fromits "internal" aldimine position, in which the A face lies againstthe 3 sheet, to one in which the A face is exposed. This ringreorientation is compatible with the exposure of the re side ofthe "internal" aldimine structure in AATase (48), and also intyrosine decarboxylase (61), and of the si side of "external"aldimine coenzyme-substrate intermediates in several B-6-dependent enzymes (38). Rotatory movement of the coenzymein reaction intermediates has been observed during single-crystal linear dichroism studies (62) and is demanded by thegeometric constraints placed on the substrate and coenzymein the present model. The second rearrangement is a bulkmotion of the small domain towards the active center. This wasobserved in crystals of apoenzyme into which N-(5'-phospho-pyridoxyl)-L-aspartate, a coenzyme-substrate analogue, haddiffused (23). The bulk movement is probably related to the"syncatalytic" increases in reactivity observed for Cys-390 (5,6) in cAATase and for Cys-166 (7) in mAATase. These residueslie beside the boundary of the small domain, where variationsin accessibility of residues could be expected.

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This work is part of an investigation on structure and function ofmitochondrial aspartate aminotransferase, a joint project with ProfessorP. Christen and colleagues at the University of Zurich. We gratefullyacknowledge the critical reading of this manuscript by Professor J.Kirsch. This work was supported in part by grant 3.415-0.78 from theSwiss National Science Foundation. The necessary computations wereperformed on a UNIVAC 1108 of Sandoz AG under a contract withthe University of Basel.

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