acyl-coa oxidase 1 from arabidopsis thaliana. structure of a key enzyme in plant lipid metabolism

doi:10.1016/j.jmb.2004.10.062 J. Mol. Biol. (2005) 345, 487–500

Acyl-CoA Oxidase 1 from Arabidopsis thaliana.Structure of a Key Enzyme in Plant Lipid Metabolism

Lise Pedersen and Anette Henriksen*

Carlsberg Laboratory, GamleCarlsberg Vej 10, DK-2500Valby, Denmark

0022-2836/$ - see front matter q 2004 E

Abbreviations used: ACX, acyl-Cacyl-CoA dehydrogenase; MCAD, mr.m.s.d., root-mean-square deviationBank; ASA, accessible surface area.E-mail address of the correspond

[email protected]

The peroxisomal acyl-CoA oxidase family plays an essential role in lipidmetabolism by catalyzing the conversion of acyl-CoA into trans-2-enoyl-CoA during fatty acid b-oxidation. Here, we report the X-ray structure ofthe FAD-containing Arabidopsis thaliana acyl-CoA oxidase 1 (ACX1), thefirst three-dimensional structure of a plant acyl-CoA oxidase. Like otheracyl-CoA oxidases, the enzyme is a dimer and it has a fold resembling thatof mammalian acyl-CoA oxidase. A comparative analysis includingmammalian acyl-CoA oxidase and the related tetrameric mitochondrialacyl-CoA dehydrogenases reveals a substrate-binding architecture thatexplains the observed preference for long-chained, mono-unsaturatedsubstrates in ACX1. Two anions are found at the ACX1 dimer interface andfor the first time the presence of a disulfide bridge in a peroxisomal proteinhas been observed. The functional differences between the peroxisomalacyl-CoA oxidases and the mitochondrial acyl-CoA dehydrogenases areattributed to structural differences in the FAD environments.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: acyl-CoA oxidase; b-oxidation; peroxisomes; fatty acid oxi-dation; FAD
*Corresponding author
Introduction

Fatty acids are essential molecules for life, servingas energy storage, signal molecules, and precursorsfor steroid hormones and as structural molecules inbiomembranes. The degradation of fatty acids is acomplex process involving many enzymes fromdifferent cellular compartments. The core of perox-isomal fatty acid catabolism is the b-oxidation cycle,in which fatty acid thioesters are oxidized in the b-position followed by removal of a C2 unit.

1,2

Acyl-CoA oxidase 1 (ACX1) is one out of fourdifferent acyl-CoA oxidases from the model oil-seedplant Arabidopsis thaliana, which catalyze theconversion of acyl-CoA into trans-2-enoyl-CoA asthe first and rate-limiting step of peroxisomalb-oxidation.3 The members of the ACX familyhave different chain-length specificity for the acyl-CoA substrate, but together they encompass theoxidation of the full range of acyl chains present invivo.4–7 ACX1 has broad substrate specificity

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oA oxidase; ACAD,edium chain ACAD;; PDB, Protein Data

ing author:

ranging from medium to long-chain acyl-CoAs,with maximum activity towards C14-CoA. The Km

value with this substrate is 5.3 mM.3 The activity ofACX1 on the long-chain mono-unsaturated sub-strates C16:1-CoA and C18:1-CoA have beenobserved to be 40% higher than with the corre-sponding saturated substrates. A similar mono-unsaturated preference has been observed for thelong-chain-specific ACX2.3

Peroxisomal b-oxidation is by far the mostpredominant fatty acid catabolic pathway in plants,especially during germination.8 The product of thepathway is acetyl-CoA, which is ultimately used asa carbon skeleton for gluconeogenesis.9 A functionalperoxisomal b-oxidation is an absolute requirementfor germination and early seedling development inoil-seed plants,10 and plays an essential role inapoptotic processes.11 The localization of all theenzymes from a single pathway in one organellehas given rise to the idea of peroxisomal b-oxidationas a highly efficient structural entity, enablingchanneling of metabolites.12 That acyl-CoA oxi-dases are imported into peroxisomes as fullyfolded, co-factor-containing multimeric complexesin the fungus Yarrowia lipolytica13 supports the ideaof ACX1 being part of a peroxisomal b-oxidationmetabolon in vivo.While plants and several fungi are likely to be

d.

488 Structure of Plant Acyl-CoA Oxidase 1

able to completely degrade fatty acids within theperoxisomes, mammalian lipid breakdown isperformed as a combined action by two distinctb-oxidation pathways in mitochondria and peroxi-somes.1,2 The substrates oxidized in peroxisomesand mitochondria partly overlap; mitochondria arecapable of oxidizing medium and long-chain mono-and dicarboxylic fatty acids, 2-methyl-branchedfatty acids and prostaglandins, while the peroxi-somes are devoted to the task of oxidizing verylong-chain fatty acids and bile acid intermediates.14

ACX deficiency and disorder in the peroxisomalb-oxidation pathway inmammals give rise to severepathological conditions like pseudo-Zellweger syn-drome,15 d-bifunctional protein deficiency16 andnon-alcoholic steatohepatitis.17

ACXs need the co-factor flavin adenine dinucleo-tide (FAD) for conversion of fatty acyl-CoA intotrans-2-enoyl-CoAs. Catalysis is a two-step reaction.The first step is an oxidative introduction of adouble bond between the substrates Ca and Cb

atoms resulting in the production of trans-2-enoyl-CoA and reduction of the FAD co-factor to itshydroquinone form (Scheme 1A). In the second(oxidative) half-reaction (Scheme 1B), FADHK

donates a hydride ion to molecular oxygen, result-ing in the formation of H2O2.

In general, ACXs have low (12% to 22%) levels ofsequence identity with the acyl-CoA dehydrogen-ase (ACAD) family, catalyzing the analogousb-oxidation step in mitochondria. ACADs are alsoflavoenzymes and like ACXs they perform an a,b-

Scheme 1.

dehydrogenation of acyl-CoA into trans-enoyl-CoA.The two enzyme families differ, however, in theoxidative half-reaction. Where the reduced ACXsare re-oxidized by molecular oxygen generatingH2O2, ACADs are re-oxidized by the electron-transferring flavoprotein from the electron trans-port chain. In this sense the peroxisomal pathwaycan be regarded as catalyst of anabolic processesproviding building blocks for gluconeogenesis,while the mitochondrial pathway is catabolic andATP generating. Which of these pathways thatprovide acetyl-CoA for biosynthesis, producemodified acyl-CoAs or remove free fatty acidsoriginating from protein and membrane lipid turn-over is unknown.

Three-dimensional structures of six ACADs areknown. These include medium-chain ACAD(MCAD) from pig and human,18,19 short-chainACAD from rat (SCAD),20 human isovaleryl-CoAdehydrogenase (i3VD) and isobutyryl-CoA dehy-drogenase (iBD)21,22 and buturyl-CoA dehydrogen-ase (BUC) from Megasphaera elsdenii.23 PorcineMCAD is by far the most extensively studiedenzyme and its structure is used as referencestructure for ACADs here. The level of sequenceidentity between ACX1 and MCAD is 9%. Amechanism for the reductive half-reaction wasproposed based upon mechanistic and structuralstudies of MCAD. The mechanism involves abstrac-tion of the acyl-substrate’s a-proton by a glutamateresidue acting as a catalytic base and the concertedtransfer of the b-hydrogen atom as a hydride ion to

Structure of Plant Acyl-CoA Oxidase 1 489

the N5 position of the FAD isoalloxazine ring(Scheme 1A).18,24–28 Structural and mechanisticstudies on peroxisomal acyl-CoA oxidases havebeen scarce and only recently the structure ofperoxisomal rat acyl-CoA oxidase-II (ACO-II) wasreported.29

Here, we report the X-ray structure of ACX1,catalyzing the first committed step in peroxisomalb-oxidation. In general, little is known about theoxidative half-reaction mechanism in FAD-contain-ing oxidoreductases utilizing molecular oxygen asthe final electron acceptor. Comparison of ACXsand ACADs provides a unique opportunity forstudying differences in oxygen reactivity. Being thefirst structure of a plant ACX and only the secondperoxisomal acyl-CoA oxidase structure, the ACX1structure provides insight into the structural basisfor the variety in substrate preferences displayed bythese enzymes.

Results and Discussion

The structure of ACX1

ACX1 is a dimer with approximate dimensions106 A!70 A!65 A. Each monomer is composed offour domains: an N-terminal a-helical domain(residue 29 to residue 130), a b-sheet domain(residue 131 to residue 272) and a C-terminal a-helical domain consisting of two helical sub-domains named Ca1 (residue 273 to residue 446)and Ca2 (residue 1 to residue 28 and residue 447 toresidue 655) (Figure 1). The related ACO-II struc-ture (RCSB Protein Data Bank entry 1IS2)29 used tosolve the ACX1 structure by the molecular replace-ment technique can be superimposed on ACX1witha root-mean-square deviation (r.m.s.d.) of 1.6 Ausing 1162 of the 1316 Ca atoms.30 Residues notsuperimposable are found in the loop sectionsconnecting the helices in the Na-domain, in helixaF, in the 310-helix in the Nb-domain, in the N-terminal of helix aG, in the loop between aG andaH, in the C-terminal of helix aK and in the loopbetween helix aL and aM (Figures 2 and 3). Whilethe overall fold and topology of ACX1 resemblesACO-II there are a substantial number of structuraldifferences, which makes ACX1 unique. Thesefeatures include a change in the relative orientationof helixes in the Na domain, a different FADembedding, a well-defined aG-helix in the Ca1-domain of ACX1 and the presence of an anion sitein ACX1.

Na domain

The N-terminal a-helical domain is composed offive a-helices (aA, aC, aD, aE, and aF) (Figures 2and 3) and has a fold similar to the correspondingdomain in ACO-II. Some core structural elements inthe Na domain are shared with ACADs. These arehelix aA, aD, aE, aF and the C-terminal part of helixaC. aB is unique to ACADs, while aC has a three-

turn N-terminal extension in oxidases. A super-position of ACX1 and MCAD shows that althoughmost structural elements are conserved, theirrelative orientation is not the same (Figure 4). Thatis, the Na domain of ACX1 is rotated by approxi-mately 138 relative to its orientation in MCAD.Overall, the same relative rotation was observed inACO-II.29 In particular, helix aD is translated 8 Aaway from helix G and the Ca1 domain in theoxidases, most likely caused by a very short spacersequence between aC and aD in these enzymes. Anapproximately 16 residue long stretch with irregu-lar secondary structure separates the aC and aDhelices in ACADs.A Glu-Glu motif is conserved in aD in ACADs.

These glutamate residues participate in salt-bridgesto helices aA and aH, and their conservationsuggests that they play a role for the structuralintegrity of ACADs. The Glu-Glu motif is notconserved in ACXs. aD contributes to the expectedfatty acyl binding site and its position in oxidasesapparently makes the fatty acyl binding site muchmore solvent-accessible and less sterically restrictedthan the corresponding binding site in ACADs(Figure 5). The relative position of aD, aE and aFand the loop connecting aD and aE varies withinthe oxidase family as well. These variations haveconsequence for the volume and shape of the distalpart of the acyl-binding pocket, which is severalangstrøm units wider in ACX1 than in ACO-II.

Nb domain

The b-domain is composed of a three-strandedantiparallel b-sheet (strands 3b, 6 and 7a) connectedto a six-stranded two-layered b-sandwich or cup-like structure (strands 1, 2, 3a, 4, 5 and 7b) with ab-hairpin (strands 8 and 9) forming the bottom ofthe cup (Figure 2). Because of an extension ofstrands 4 and 5, these strands are part of both facesof the b-sandwich in oxidases, whereas they onlycontribute to the FAD/Na domain interacting facein ACADs. A small bulge in b-strands 3 and 7 isintroduced in the bent region of the Nb domain inACXs. This bulge is not found in ACADs. The bulgeis surface-exposed and residues from the bulgemake interactions to the aU helix from the othersubunit thereby stabilizing dimer formation. Theb-hairpin (strands 8 and 9) is also unique to ACXsand interacts with the ACX extensions in strands 4and 5 (Figure 2).

Ca domains

The C-terminal part of ACX1 is divided into twoa-helical domains. There has been some inconsis-tency in previous helix nomenclature, but ACX1helix nomenclature is based on an extension of thatdescribed by Kim & Miura.31 Ca1 is composed offive a-helices (aG to aK) of which helices aG, aH, aI,and aJ form a four-helix bundle. A similar helicalbundle is found in ACADs and most of thecatalytically important residues belong to this

Figure 1. Stereo view of ACX1. A, Subunit A is shown in gray while subunit B is colored according to domainstructure: Na domain in blue, Nb domain in purple, Ca1 domain in green and the Ca2 domain in orange. The two FADmolecules are represented in ball-and-stick. Identified anions are represented as green spheres. The Ca2C is representedby a red sphere. B, A Ca trace of ACX1 colored as for (a), with every 50th residue numbered and with the C8-CoAsubstrate from the MCAD:C8-CoA complex superimposed. C, Stereo view of the subunit organization in tetramericMCAD. Each subunit is shown in its own color; although the Ca domain of the green subunit is colored light green toemphasize that its position in the tetrameric ACAD arrangement resembles the position of the Ca2 domain in the dimericACX structures. The orientation of the blue and purple subunit corresponds to the orientation of the correspondingsubunits in the ACX1 dimer in A.


domain. This is true for FAD-binding, fatty acyl-binding and active-site residues. The N-terminalpart of helix aG (poorly defined in ACO-II29) has anunusually long32 three-turn 310-helical structure inACX1 and a less curved shape than the

corresponding helix in ACADs. The differentcurvature causes the N-terminal of helix aG to bedisplaced by approximately 10 A. We expect thispart of the aG-helix to be involved in CoA bindingby analogy to the MCAD:C8-CoA complex,18 the

Figure 2. Domain structure ofACX1 and MCAD. Domains havethe same colors as for Figure 1A.Secondary structural elements arelabeled. A, The ACX1 subunit inthe same orientation as the right-hand subunit in Figure 1A. B, Thesame subunit after a 908 rotationabout the vertical axis. C, MCADsubunit in the same orientation asthe right-hand subunit of ACX1 inFigure 1A. D, The same subunitafter a 908 rotation about thevertical axis.


MCAD:3S-C8-CoA complex,33 the IVH:methacry-lyl-CoA complex22 and the SCAD:acetoacetyl-CoAcomplex.20 It is possible that fatty acyl-CoA bindingintroduces a regularization of the N-terminal part ofaG, but the induction of an ACAD-like packing ofaG is not possible in ACX1. The b-hairpin segmentforming the bottom of the b-cup in ACXs wouldinterfere with such packing. It is more likely that theadenosine moiety of the fatty acyl-CoA moleculeadapts a different orientation in ACX1.

An a-helix (helix aL) is included in the ACX1 Ca2

domain in addition to the helices defined in theACO-II model. The aL helix can be regarded as alinker to the Ca2 sub-domain consisting of twohelices from the N-terminal part of the sequence (aaand ab) and the 11 C-terminal helices (aN throughaV). A four-helix bundle is formed by helices aM,aO, aP and aR. Helices aa/ab, and aS/aTconstitutethe interface between the Ca2 and the Na domains.The last two helices (aU and aV) generate a longarm on top of the molecule extending to the Na andNb domains of the other subunit.

The Ca2 domain contributes to the embedding ofthe FAD adenine moiety at the dimer interface.Nothing reminiscent of or substitute for the Ca2 sub-domain is found in ACAD monomers (Figure 1C).The Ca2 sub-domain has previously been argued toplay the role of protecting helix-bundle Ca1 againstsolvent and preventing tetramer formation.29 Fromthe functional tetrameric ACADs arrangement(Figure 1C), it is obvious, however, that each Ca1

domain in these enzymes can play a functional rolein two subunits. We suggest the ACX Ca2 domain tobe a result of a domain duplication event, substitut-ing for the tetrameric ACAD arrangement by theintroduction of an extra Ca domain only andthereby allowing the formation of a functionalACX dimer (Figure 1A and C). A robust packing

of this minimal structure must be expected to be anadvantage if the ACX is to be transported over theperoxisomal membrane as a folded entity or even aspart of a metabolon. An additional function of theCa2 domain could be in mediating contact betweensubunits in the oxidase metabolon.A disulfide bridge is formed between Cys467

from the loop between helices aL and aM, andCys576 from the loop between helix aQ and aR. Thepresence of cysteine residues in this region is notcharacteristic for members of the ACX family, butcorresponding cysteine residues are found in thesequence of a putative acyl-CoA oxidase fromHordeum vulgare. A disulfide bridge at anotherlocation has been observed in isovaleryl-CoAdehydrogenase.21 The oxidative environment ofthe peroxisomes suggests that the cysteine residuesmost likely exist in their oxidized form in vivo. Toour knowledge, this is the first disulfide bridgeobserved in a peroxisomal protein. Recently, theformation of an intramolecular disulfide bridge as areaction intermediate was characterized in mam-malian peroxiredoxin.34

Domain interaction

Nakajima and co-workers have taken theobserved relative rotation of the Na domain as anexpression of flexibility in the N-terminal region ofACXs.29 Although the Na/Ca1 domain interface isdominated by polar and ionic interactions it definesthe fatty acyl binding part of the binding pocket forthe fatty acyl-CoA substrate and it was suggestedthat the Na domain could function as a lid closingupon substrate binding. An r.m.s.d. value of4.2 A from the superposition of Ca positions in theNa domains of ACX1 and ACO-II could indicateflexibility of the domain. However, the parts of the

Figure 3. Structural alignment of A. thaliana ACX1, rat ACO-II and pig MCAD. Secondary structural elements areincluded with a-helices in black and 310-helices in gray. Parts of the ACO-II structure with unassigned atomiccoordinates are marked as white letters on a gray background. The active site glutamate residue is indicated by a filledtriangle, while the glycine residues responsible for oxygen accessibility are marked as white letters on a blackbackground. Residues interacting with FAD have black letters on a gray background. The residues involved in the ACX1anion site are indicated by circles and in the Ca2C site by open triangles. The alignment was prepared using theprograms CE54 and ALSCRIPT.55

† http://wolf.bms.umist.ac.uk/naccess


domain with highest r.m.s.d. values are the aE andaF helices and not the aA, aC and aD helices asexpected from the overall ACAD/ACX superposi-tion. It should be noted that the observed orien-tation of the dimer-spanning helical arm in Ca2

would interfere with a re-orientation of the Na

domain. Also, the atomic B-values in the Na domainand its linker segments are not significantly higherthan in the rest of the structure. From theseobservations, we find it unlikely that the Na domainhas a lid-closing function in ACXs, and we suggestthat the Na/Nb interface is more susceptible tostructural rearrangements upon substrate bindingthan the position of the aD helix.

Dimer interface

Consistent with the observation that ACX1 formsa dimer in the crystal structure, a molecular mass ofapproximately 140 kDa is estimated by gel-filtrationexperiments (data not shown). The extensive dimerinterface buries 5900 A2 of the accessible surfacearea (calculated with NACCESS†), mainly involvinghelices aC, aJ, aU and aVand one side of the b-cup.The dimer interface covers 20% of the totalaccessible surface area.

http://wolf.bms.umist.ac.uk/naccess

Figure 4. Superposition of the N-

terminal domains from ACX1 andMCAD. A complete ACX1 subunitis included in the Figure (white)while only the two N-terminalMCAD domains are included(black). Helices in the Na domainare labeled. The superposition wasmade in O.30 It is based on theposition of the active site glutamateresidue, but extended to include thewhole molecule with the lsq_impoption. A ball-and-stick model ofFAD from the ACX1 structure isincluded (white) as well as the C8-CoA substrate (black) from theMCAD structure.18


Two spherical 7speakswere observed in a jFoKFcjresidual electron density map. The peaks werepositioned at the dimer interface, 3.0–3.2 A from thebackbone nitrogen atoms of Gly230, Gln231,Gly230*, Gln231* and two water molecules (Figure6). Given the electron density level and the fact thatall neighboring atoms are potential hydrogen bonddonors, the atoms contributing to the electrondensity are likely to be anions of a higher atomicnumber than oxygen. The inclusion of chloride ionsin themolecular structure at these positions results inless than 3s jFoKFcj electron density and atomicanion B-values of 19 A2 and 43 A2. Chloride ionsare added to the present crystal in the K2PtCl4 soak,but similar electron density levels in these positionsare observed in a lower-resolution data from acrystal not soaked in K2PtCl4 (data not shown).

The FAD co-factor is found at the dimer interfaceand dimer formation is essential to FAD binding.The anion binding turn appears to be conservedamong the group of long-chain-specific plant ACXs(data not shown), but it is absent from other ACXs(e.g. ACO-II) and ACADs. Placed on the dimerinterface, the anions could play a regulatory role inthe dimerization event in a sub-group of ACXs.

The subunits

The overall variation in subunit structure is low,giving an r.m.s.d. of Ca positions of 0.5 A. Onecalcium ion per dimer is located between the side-chains of Glu9 and Asn12* (Figure 1). The calciumion probably represents an artifact from crystal-lization, where 0.2 M calcium acetate was includedin the buffer. The largest deviations in Ca positionsbetween subunits are found in the solvent-exposedloops in the Ca1 domain. As this domain is involvedin fatty acyl binding, it is likely that some of thestructural variation in this domain is caused by theabsence of substrate.

FAD binding

The FAD molecule is located in a crevice between

the Nb and the Ca1 domain of one subunit and theCa1 domain of a neighboring subunit (Figure 7). Itis bound via several hydrogen bonds and hydro-phobic interactions. The FAD adapts an extendedshape, as in the crystal structures of ACO-II andACADs. The si-face of the FAD ring is orientatedtowards the Nb-domain making stacking inter-actions with the conserved Trp175 and Pro178.One of the phosphate groups interacts with a P-loop(residues 399 to 404), having the primary sequenceCGGHGY and containing the “GG” doubletdescribed as a consensus motif in several flavopro-tein families.36 The other phosphate group interactswith Arg310, another conserved residue in theACAD superfamily.The 2 0 and 3 0-hydroxyl groups of the ribose are

connected via hydrogen bonds to Gln330 O3 andAsp426 Od1 and Od2. Neither Gln330 nor Asp426 isconserved in the superfamily. The ribityl chain ofFAD is in the gauche conformation with respect tothe 2 0 and 3 0-OH. This conformation is energeticallyless favorable than the commonly observed transconformation and has not previously been observedin the ACAD superfamily. A network of inter-molecular hydrogen bonds stabilizes this confor-mation. The hydrogen bond (2.7 A) between theribityl 4 0-OH group and Thr422 Og is unique forACX1. In ACADs, the 2 0-OH group contributes toan oxyanion hole for the carbonyl oxygen atom ofthe thioester substrate together with the backbonenitrogen atom of the catalytic base. The oxyanionhole promotes the acidification of the Ca proton.18

When no fatty acyl-CoA substrate is bound inACX1, the 2 0-OH group of the ribityl chain canmake a hydrogen bond to FAD N1, Gln137 N3 andThr138 Og via a water molecule, while 3 0-OH makesa hydrogen bond to the side-chain of Thr138 (Figure7). A superposition of ACX1 with the MCAD:C8-CoA complex shows that the 2 0-OH group can havesimilar roles in the two enzymes.Transfer of electrons to the flavin ring during

catalysis produces a reduced anionic flavin species,which has negative charge delocalized on thepyrimidine ring. The hydrogen bonds between

Figure 5. Fatty acid-binding pockets of ACX1 andMCAD. a, Surface-accessible area of the fatty acyl-CoAbinding pocket in ACX1. The C8-CoA molecule shown ingray originates from a superposition with the MCAD:C8-CoA complex. The flavin ring of the FADmolecule is seenin the lower right corner of the pocket. b, Surface-accessible area of the corresponding pocket in theMCAD:C8-CoA complex. The program msms56 wasused to generate the solvent-accessible surfaces and theFigure was prepared with the program DINO (http://www.bioz.unibas.ch/~xray/dino).


FAD O4 and the backbone nitrogen atom of Gly177and between FAD N3 and the backbone oxygenatom of Tyr135 increase electron delocalization.Similar backbone interactions to FADO4 andN3 arefound in the ACAD structures, but not in ACO-II.The negative charge on the ACX1 FAD ring can bestabilized by an oxyanion hole formed by thebackbone amide groups of Gln137 and Thr138surrounding FAD O2 and by a hydrogen bondbetween FAD N1 and Thr138 Og.

The oxyanion hole for FAD O2 and the threoninehydrogen bond are also observed in all ACAD

structures and similar flavin interactions areobserved in flavocytochrome b2 and glycolateoxidase,36,37 where a lysine Nz is located close tothe FAD N1/O2 atoms. In contrast to the situationin ACX1 and ACADs, the only hydrogen bond(3.1 A) to the FAD O2 in ACO-II is to the side-chainof Thr139 Og (positional equivalent of ACX1Thr138) (Figure 7).

A hydrogen bond from the FAD N5 atom to ahydrogen bond donor is a recurrent feature in mostoxidoreductase flavoenzymes.38 In ACX1, N5makes hydrogen bonds to Tyr234 Oh and Asn239Nd2 through water molecules. Asn239 is a con-served residue among ACX sequences, whileTyr234 is conserved among long-chain-specificacyl-CoA oxidases. In contrast, FAD N5 is notinvolved in any hydrogen bonds in ACO-II, whilein all ACAD structures it is hydrogen bonded to aThr Og atom. Two Gly residues proposed to providebetter access for molecular oxygen replace this Thrresidue and the following Asp residue in ACXs.

A low number of hydrogen bonds between theflavin ring and the polypeptide chain have beensuggested to facilitate access of molecular oxygen tothe FAD ring in ACO-II.29 As the number ofhydrogen bonds between the flavin ring and theprotein in ACX1 equals that in ACADs, this seemsnot to be a general feature in the ACX family (Figure7). A calculation of the accessible surface area (ASA)of the flavin ring in ACX1, ACO-II andMCAD givesvalues of 23 A2, 34 A2 and 23 A2, respectively. Still,ACX1 and ACO-II are comparable if the ASA ofsome key FAD atoms is considered. N5 in ACX1and ACO-II have ASA values of 5.6 A2 and 5.2 A2

while the ASA values for C4a are 2.7 A2 and 2.6 A2,respectively. The corresponding atoms in MCADhave values of 2.4 A2 and 1.9 A2. Molecular oxygenactivation has been shown to proceed via formationof remarkably stable covalent C4a-flavin hydroper-oxides in flavin-dependent mono-oxygenases.39

This mechanism has been considered the commonroute for oxidation of reduced flavin in all oxido-reductase flavoenzymes. The larger ASA of the N5/C4a atoms in ACX1 and ACO-II supports thissuggestion.

The larger flavin ASA of ACO-II compared toACX1 co-segregates with a lower number ofhydrogen bonds (Figure 8), and it appears thatthere is a certain degree of flexibility in the positionof the flavin ring in ACXs when no substrate isbound. However, the comparable FAD environ-ments in ACADs and ACX1 do not suggest that co-factor dynamics39,40 should have a more pro-nounced role in the oxidative half-reaction inACXs than in ACADs.

Active site architecture and substrate bindingpocket

The position of the substrate-binding pocket ofACX1 is similar to that observed in ACO-II andACAD:substrate complexes.18,20,22,33 The fatty acyl-binding pocket is formed by a-helices aD, aE, aG,

http://www.bioz.unibas.ch/~xray/dino

http://www.bioz.unibas.ch/~xray/dino

Figure 6. ACX1 anion site. The site is located at the dimer interface and involves residues from the Nb domain. Theresidues contributing to the site are conserved within a sub-group of ACXs. Dotted black lines indicate hydrogen bondsand anion interactions. Water molecules are included as gray spheres.


aH and aJ. The residues lining the pocket, definedby a superposition with the MCAD:C8-CoA com-plex are approximately 50% identical in ACX1 andACO-II, and overall the pocket is not hydrophobic.

Several conserved residues like Arg436 arelocated near the entrance of the fatty acyl-bindingpocket serving as potential interaction site for theCoA moiety of the substrate. Water molecules linethe part of the pocket where the pantetheine moiety

Figure 7. FAD binding site in ACX1. Green residues originsubunit 2. The FAD co-factor is in orange. Possible hydrogen

is expected to bind, while the expected adenosine-binding site appears more flexible with highB-values and no well-resolved water molecules.The high B-value loops are the loop connecting theNb-domain with the aG-helix, and the loop con-necting helices aH and aI including the last turn inhelix aH and the first turn in helix aI. The observedflexibility in the adenosine-binding site agrees wellwith the medium to long-chain specificity of ACX1.

ate from subunit 1, while white residues originate frombonds are included as dotted black lines.

Figure 8. Superposition of the FAD and FAD environment of ACX1 and ACO-II. The polypeptide surrounding FAD isrepresented as a black coil in ACX1 and as a gray coil in ACO-II. The superposition was made in O.30 Ball-and-stickmodels of FAD from ACX1 and ACO-II are shown in black and gray, respectively. FAD interacting residues from ACX1are included in the Figure as ball-and-stick models. The superposition is based on the ACX1 residues Thr138, Ser144,Trp175, Tyr423, and Glu424.


To obtain this specificity, the acyl chain mustcontribute significantly to the binding energy,while a too well fitting framework for CoAembedding is not an advantage.

Only a limited number of amino acid residues areconserved in the fatty acyl-binding pocket of ACXs.The conserved residues include Tyr135 (Phe or Tyr),Leu101 (Leu or Val), Met105 (Met or Leu), andTyr423 (Tyr, Trp or Phe), all found in close proximityto FAD and likely to interact with the Ca end of theacyl-CoA substrate.

ACX1 is active on fatty acyl-CoA with a chainlength between C10 and C18, having a Km value forthe reaction with the preferred substrate C14-CoA of5.3 mM.3 Rat ACO-II has been reported to have asimilar substrate profile.41 Still, the fatty acyl-binding pockets have different size and shape. InACX1, the length of the pocket is 18 A measured asthe atom-to-atom distance from the position of theCa atom in the superimposed MCAD:C8-CoAcomplex to the carboxyl group of Asp94 at thebottom of the pocket. The width of the pocket is12 A at the midpoint of this line. Both fatty acyl-binding pockets broaden towards the distal end andare solvent-exposed (Figure 5). Well-resolved watermolecules line the distal half of the ACX1 pocket,while the FAD vicinity has no well-ordered watermolecules. The B-value distribution and electrondensity quality does not indicate large flexibility inthe structural elements contributing to the pocket.Rather, the water molecule distribution seems toreflect a more hydrophobic environment in the FADvicinity.

At the bottom of the ACX1 pocket, Asp94 andArg298 form a salt-bridge. The position of the salt-bridge is in good agreement with the observed C14

chain-length preference of ACX1. A two residueinsertion in the loop preceding aE in ACO-II

displaces the polypeptide chain by 3.4 A in thisenzyme and cuts off the fatty acyl-binding pocket.Sequence alignments with A. thaliana ACX23 andACX35,6 show that these enzymes have an insert inthe same area. That ACO-II, having the samesubstrate profile for saturated substrates as ACX1have a less spacious distal pocket, infer that acylchain conformation and enzyme interactions are notthe same in the two enzymes. It is worth noticingthat the differing substrate preferences of shortchain and isovaleryl-CoA dehydrogenases aredefined by insertions in aE.20,31 This helix andadjacent residues constitute one of the least con-served areas in ACX and ACAD structures, and thedefinition of substrate specificity by introduction ofmodifications in this area could be a featurecharacteristic of the ACAD superfamily.

ACX1 has been observed to prefer mono-unsatu-rated over saturated fatty acyl substrates for chainslonger than C14.

3 The introduction of a double bondat the C9 position in palmitoleic and oleic acid willin the cis form introduce a kink in the acyl chain andin both the cis and trans form reduce flexibility. Themono-unsaturated substrate preference is sustainedby both factors. The wide ACX1 pocket has room fora kinked substrate and the constrained substrateconformation will contribute to complex formationby lowering the entropic barrier.

Jasmonic acid is a signal molecule in plantwound-activated defense.43 Part of jasmonic acidsynthesis takes place in the peroxisomes, wherethree steps of b-oxidation complete the synthesis.ACX1 is the only Arabidopsis ACX gene induced bymechanical damage and jasmonic acid, andexpression of ACX1 antisense mRNA results inreduced jasmonic acid synthesis.43 That ACX1, incontrast to ACX2 and ACX3 participates in jasmo-nic acid production is in agreement with the


observed wide fatty acyl-binding pocket of ACX1and the loop insertions preceding the aE helixfound in the ACX2 and ACX3 sequences.

The solvent accessibility of the ACXs fatty acyl-binding pocket could be linked to the fate of thereducing equivalents in this system. ACADs passthe equivalents to another protein, but ACXsproduce the small molecular species H2O2. H2O2

has the potential to cause oxidative damage to theenzyme and a fast exit of the generated H2O2 isadvantageous to preserve an active enzyme. Thehydrophobicity of the immediate FAD vicinitycould be another factor contributing to a fastexpulsion of H2O2.

The catalytic ACX1 Glu424 (Scheme 1) is posi-tioned in a flexible turn between the very well-defined helices aJ and aK. It has a 2jFoKFcj electrondensity level of 0.6s at the Cg atom. The rest of theresidue has a 2jFoKFcj electron density level above1.0s. Only one water molecule has been assignedwithin hydrogen bonding distance of Glu424.Glu424 superimposes well with the catalytic basein ACADs. It is positioned over the re-face of theflavin ring with the carboxylate group positionedideally for Ca proton abstraction. In ACO-II, theglutamate is too far from the expected Ca atomposition for de-protonation to occur. From thecrystal structure of ACX1 and available mechanisticstudies on ACADs,44 the following catalytic mech-anism of ACX1 is proposed. The acyl-CoA substratebinds to the substrate-binding pocket therebypositioning the Ca–Cb bond between the catalyticGlu424 and the flavin ring. Glu424 abstracts the Ca

proton from the substrate while the ribityl 2 0-OHgroup and the backbone amide from Glu424stabilizes the thioester carbonyl group duringcatalysis. No structural rearrangements are neededfor this reaction to occur. Simultaneous transfer of ahydride ion to FAD N5 reduces FAD to a twoelectron reduced semi-quinone species. The nega-tive charge that develops on the flavin ring duringcatalysis is stabilized by the amide groups ofGln137, Thr138, Gly177 and through a hydrogenbond between FAD N1 and Thr138 Og. Release ofsubstrate is followed by access for molecularoxygen to the solvent-exposed C4a atom on theflavin ring. Two residues near the flavin ring areconserved in the ACX family. These are Asn239 andLeu101. The residues are not conserved in ACADs.Both residues are in close proximity of C4a,suggesting that they play a role in the oxidativehalf-reaction.

Conclusion

The principal difference between peroxisomalACXs and mitochondrial ACADs is the enhancedreactivity towards molecular oxygen of ACXs. Thereplacement of a Thr and an Asn residue close to theFAD N5-C4a atoms in ACADs with Gly residues inACXs is likely to provide access of molecularoxygen to the co-factor. ACX1 displays a more

open fatty acyl-CoA-binding pocket compared tothe active sites in ACADs, at least in the absence offatty acyl-CoA substrate. No structural obser-vations indicate a larger conformational changeupon substrate binding and the spacious andsolvent-accessible fatty acyl-binding pocket andactive site are probably general features of ACXsallowing fast exit of H2O2. However, the morespacious fatty acyl-binding pocket does not effectthe number of hydrogen bonds between FAD andACX1, which is similar to the number foundbetween FAD and ACADs. The residues respon-sible for substrate specificity in ACX1 has beenmapped to the loop preceding aE. Modifications inthe aE area appear to be a general feature fordefinition of substrate specificity among ACADs.

Materials and Methods

Protein expression and purification

Recombinant ACX1 was expressed and purified asdescribed.45 In short, recombinant ACX1was produced inEscherichia coli BL21(DE3) Gold cells (Stratagene) with anon-cleavable C-terminal 6!histidine tag. ACX1 waspurified on a HiTrap Ni-chelating column (AmershamPharmacia) followed by size-exclusion chromatographyon a Superdex 75 column. Activity of ACX1 wasmeasured at 20 8C by the peroxidase-coupled methoddescribed by Gerhardt46 using 25 mM pristanoyl-CoA assubstrate. The presence of the 6!His-tag does notinterfere with the activity of the protein.45

Crystallization and data collection

Purified recombinant ACX1 was dialyzed against20 mM Hepes (pH 7.0), 10 mM FAD and concentrated to8 mg/ml using a Centricon 30 (Millipore). Crystals weregrown at 20 8C by the hanging-drop, vapor-diffusionmethod by mixing 2 ml of reservoir solution with 2 ml ofprotein solution. The reservoir solution contained 0.2 Mcalcium acetate, 18% (w/v) polyethylene glycol (PEG)8000 and 0.1 M sodium cacoylate (pH 6.5) and crystalsgrew to maximum dimensions within two to three days.Cryo-protection of the crystals was performed by soakingof the crystals in reservoir solution containing 25% (v/v)glycerol before flash cooling in liquid N2. For heavy-atomsoaking, 20 mM K2PtCl4 was added to the crystallizationdrop 30 minutes prior to cryo-cooling. Single-crystaldiffraction data from the K2PtCl4-soaked crystal werecollected at the I711 synchrotron beam-line at Maxlab,Lund. The crystal diffracted to a resolution of 2.0 A.Reflections were indexed, integrated and scaled withMOSFLM47 and SCALA.48 The crystals belong to theorthorhombic space group P212121 with unit cell dimen-sions aZ85.6 A, bZ117.0 A and cZ131.3 A, and containstwo monomers per asymmetric unit, corresponding to aMatthews volume of 2.23 A3 DaK1. Data collectionstatistics have been reported.45 To summarize, a 95.8%complete data set was collected to a resolution of 2.0 Awith an Rmerge value of 11% and an I/sI ratio of 7.4.The Wilson B-value of the data set was 15.1 A2.

Structure determination and refinement

The structure of acyl-CoA oxidase 1 was determined by

Table 1. ACX1, model refinement statistics

Residue range included A2–A659; B2–B659No. of non-hydrogen protein atoms 10,306No. of water molecules 748Resolution (A)a 29.6–2.0 (2.1–2.0)Rfactor (%)b 20.4 (26.0)Rfree (%)c 24.9 (30.1)

r.m.s. deviation from ideal valuesBond lengths (A) 0.007Bond angles (deg.) 1.1Dihedral angles (deg.) 20.8Improper angles (deg.) 0.8

Average B values (A2)Protein 25.2Co-factor 20.0Water 31.7

Estimated coordinate error (A)d

Luzzati plot 0.28sA 0.27

Ramachandran plot (non-Gly/non-Pro)e

Most-favored regions (%) 90.4Additionally allowed regions (%) 9.1Generously allowed regions (%) 0.5Disallowed regions (%) 0.0

a Values in parentheses are for the highest-resolution shell.b R-factorZSjjFojK jFcjj=SjFoj.c Rfree was calculated with 5% of the reflections excluded from

the refinement.d This is the cross-validated coordinate error calculated in

CNS.50e Calculated in PROCHECK.53


molecular replacement using domains Nb, Ca1 and Ca2

from ACO-II29 as search model and omitting watermolecules and FAD from the structure. The ACX1 andACO-II structures have a sequence identity of 42%.Phases were determined by molecular replacement inMOLREP,49 initially using a 2.9 A data set available at thattime. Treatment of the two NCS-relatedmolecules as rigidbodies gave an initial Rfactor value of 48% in CNS.50 Anincomplete model (residues 178 to 601) was built andmodified in O.30 Further refinement in CNS using thesimulated annealing option gave an Rfactor/Rfree of 34%/42%. Phases were later extended by molecular replace-ment in the 2.0 A data set using the incomplete model assearch molecule in AMoRe.51 The new phases were usedas a starting point for automatic model building in ARP/wARP,52 by which amodel composed of 45 fragments and840 residues was produced. The electron density mapwas of excellent quality, allowing manual rebuilding ofthe model in O. The model was further improved inARP/wARP using the model improvement option.Subsequent introduction of platinum, calcium andchloride ions were judged appropriate if present inbeyond 6s in a sA-weighted jFoKFcj electron densitymap. Assignment of calcium and chloride ions was donebased upon coordination distances and nature of theligands. Addition of water and final refinement wasperformed with CNS with simulated annealing to4500 K,50 first applying strict NCS and finally with noNCS restrains applied. The level for water picking was setto 1.5s in the sA-weighted 2FoKFc electron density map.The geometry of the refined structure was checked withPROCHECK53 and appears reasonable. The two ACX1subunits in the asymmetric unit are very similar with anr.m.s.d. value between Ca positions of 0.5 A. The averageB-value is similar in the two subunits. Refinementstatistics are summarized in Table 1.

Accession numbers

The atomic coordinates and structure factors have beendeposited in the RCSB Protein Data Bank with accessionnumber 1WO7.

Acknowledgements

The Danish Natural Research Council (DAN-SYNC grant) and the European Community (Accessto Research Infrastructure Action of the ImprovingHuman Potential Program to the EMBL HamburgOutstation and Maxlab) supported access to syn-chrotron sources. We thank beam-line scientistsYngve Cerenius, beam-line I711, Maxlab, Lund andSantosh Panjikar, beam-line BW7A, EMBL, Ham-burg Outstation, who assisted in data collections.

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Edited by R. Huber

(Received 10 July 2004; received in revised form 18 October 2004; accepted 21 October 2004)

acyl-coa oxidase 1 from arabidopsis thaliana. structure of a key enzyme in plant lipid metabolism

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