Báo cáo khóa học: Acyl-CoA dehydrogenases and acyl-CoA oxidases Structural basis for mechanistic similarities and differences pot

Five members of the ACAD family are involved in fatty acid b-oxidation; these are short, medium, long and very long chain acyl-CoA dehydrogenase SCAD, MCAD, LCAD and VLCAD1, resepctively

M I N I R E V I E W

Acyl-CoA dehydrogenases and acyl-CoA oxidases

Structural basis for mechanistic similarities and differences

Jung-Ja P Kim1and Retsu Miura2

1

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA;2Department of Molecular Enzymology, Graduate School of Medical Sciences, Kumamoto University, Japan

Acyl-CoA dehydrogenases and acyl-CoA oxidases are two

closely related FAD-containing enzyme families that are

present in mitochondria and peroxisomes, respectively They

catalyze the dehydrogenation of acyl-CoA thioesters to the

corresponding trans-2-enoyl-CoA This review examines the

structure of medium chain acyl-CoA dehydrogenase, as

a representative of the dehydrogenase family, with respect

to the catalytic mechanism and its broad chain length

specificity Comparing the structures of four other acyl-CoA dehydrogenases provides further insights into the structural basis for the substrate specificity of each of these enzymes In addition, the structure of peroxisomal acyl-CoA oxidase II from rat liver is compared to that of medium chain acyl-CoA dehydrogenase, and the structural basis for their different oxidative half reactions is discussed

Introduction

There are nine known members in the acyl-CoA

dehydro-genase (ACAD) family, including the recently identified

ACAD-9, which is homologous to the very long chain

acyl-CoA dehydrogenase [1] Five members of the ACAD family

are involved in fatty acid b-oxidation; these are short,

medium, long and very long chain acyl-CoA dehydrogenase

(SCAD, MCAD, LCAD and VLCAD1, resepctively),

and ACAD-9 (hereafter referred to as VLCAD2) The

four other members are involved in amino acid oxidation

pathways; they are iso(3)valeryl-CoA dehydrogenase

(i3VD) for leucine, iso(2)valeryl-CoA dehydrogenase

(i2VD, also known as short/branched chain acyl-CoA

dehydrogenase or 2-methylbutyryl-CoA dehydrogenase) for

isoleucine, isobutyryl-CoA dehydrogenase (iBD) for valine,

and glutaryl-CoA dehydrogenase (GD) for lysine and

tryptophan With the exception of VLCADs, all of these

are soluble homotetramers with a subunit mass of

approxi-mately 43 kDa, with each subunit containing one FAD

VLCAD1 is a homodimer with a subunit mass of 73 kDa with one FAD per monomer and is bound to the matrix side

of the mitochondrial inner membrane [2] Three-dimen-sional structures of rat SCAD [3], pig and human MCAD [4,5], i3VD [6], and GD [7] have been solved As MCAD has been studied the most extensively, both biochemically and structurally, this review discusses the MCAD structure in detail as the prototype For the other ACAD family members, only those features that differ from the MCAD structure will be discussed The structure of a peroxisomal acyl-CoA oxidase (ACO) was recently determined [8] and is compared to MCAD These structures are compared with respect to the structural basis for their different oxidative half reactions ACO is reoxidized by molecular oxygen (i.e

a true oxidase), whereas MCAD is reoxidized by transfer-ring electrons to another flavoprotein, electron transfer flavoprotein (ETF) However, it should be noted that, in its ligand-free form, the reduced flavin of MCAD has relatively high oxygen reactivity compared to the product-bound form, which has virtually no oxygen reactivity [9–11] Amino acid sequences and quaternary structure The amino acid sequences of eight members of the ACAD family, from several different sources, have been deduced fromtheir cDNA sequences [12,13] With the exception of VLCAD, the sequence identities among these members range from35–45% and are evenly distributed over the entire polypeptide span, strongly suggesting that these enzymes originated from a common ancestral gene Figure 1 shows a structure-based sequence alignment of the ACADs and ACO whose three-dimensional structures have been determined A common evolutionary path is also suggested by the similarity in their overall three-dimensional structures

Like VLCAD, ACO is a homodimer with a subunit molecular mass of 75 kDa The sequence identities of the N-terminal approximately 400 residues of VLCAD and

Correspondence to J.-J P Kim, Department of Biochemistry,

Medical College of Wisconsin, Milwaukee, WI 53226, USA.

Fax: + 1 414 456 6510, Tel.: + 1 414 456 8479,

E-mail: jjkim@mcw.edu

Abbreviations: ACAD, acyl-CoA dehydrogenase; SCAD, short chain

acyl-CoA dehydrogenase; MCAD, medium chain acyl-CoA

dehydrogenase; MLCAD, medium long chain acyl-CoA

dehydrogenase; LCAD, long chain acyl-CoA dehydrogenase;

VLCAD, very long chain acyl-CoA dehydrogenase; iBD,

isobutyryl-CoA dehydrogenase; i3VD, isovaleryl-CoA

dehydro-genase; i2VD, Ôbranched chainÕ acyl-CoA dehydrodehydro-genase; GD,

glutaryl-CoA dehydrogenase; ACO, acyl-CoA oxidase; ETF, electron

transfer flavoprotein; rSCAD, rat SCAD; bSCAD, a bacterial SCAD

from Megasphaera elsdenii.

(Received 22 July 2003, revised 24 September 2003,

accepted 7 October 2003)

Fig 1 Structure-based sequence alignment of pig MCAD, rat SCAD, bacterial SCAD from M elsdenii, human i3VD and rat ACO-II a-Helices are indicated with cylinders and, b-strands with arrows Residues that are identical in all four ACADs or all five enzymes including ACO-II are shown

in a light blue box and those that are similar are shown in a pink box Residues that line the binding cavity for the acyl chain moiety of the thioester substrate are in red As the structure of ACO-II is that of the uncomplexed enzyme, its cavity lining residues are not known and therefore not marked Catalytic glutamates are marked with a white letter and red background.

ACO to the MCAD sequence (all human enzymes) are 36%

and 31%, respectively; however, the remaining C-terminal

approximately 180 residues of each, which are not present in

other ACADs, do not share any significant similarity to each

other or to any other known proteins in the data base These

facts indicate that VLCAD and ACO share a common

ancestral gene with ACADs, but have diverged and fused

with another gene to bind to the mitochondrial membrane

(VLCAD) or to become an oxidase in peroxisomes (ACO)

In addition, recently a large number of ACAD homologs

have been identified both in eukaryotes and prokaryotes;

and the numbers are likely to increase as more sequence

data becomes available Their functions range from steroid side chain cleavage [14] to antibiotic synthesis [15] and to stress response [16,17]

The three-dimensional structure of MCAD The structures of MCAD have been solved in binary complexes with substrates or inhibitors having various fatty-acyl chain lengths, as well as in the uncomplexed form [4,18] The overall polypeptide fold of a monomer of MCAD is shown in Fig 2A The monomer is composed of three structural domains of approximately equal size: the

Fig 2 Ribbon diagrams of the MCAD

structure (A) Overall polypeptide fold of an

MCAD monomer with bound octanoyl-CoA.

The FAD and the octanoyl-CoA substrate are

shown with solid balls in yellow and purple,

respectively a-Helices are labeled

alphabetic-ally and, b-strands are numbered

consecu-tively, fromthe N-terminus in both instances.

The numbers in a smaller font are residue

numbers used to help follow the polypeptide

tracing The three domains of the monomer,

the N-terminal a-helical domain, the middle

b-sheet domain, and the C-terminal a-domain

are colored in red, cyan, and green,

respect-ively (B) A ribbon diagramof a tetramer of

MCAD The molecule is viewed along one

of the three orthogonal twofold axes The view

of the green subunit is rotated  40 from

the monomer view shown in (A) The figure

was prepared using MOLSCRIPT [38] and

3 [39].

N-terminal and C-terminal domains consist mainly of

a-helices and are packed together in three-dimensions,

forming the core of the tetrameric molecule, whereas the

middle domain is composed of two orthogonal b-sheets and

lies at the surface of the molecule (Fig 2B) The tetrameric

molecule is a dimer of dimers in a tetrahedral arrangement

with an overall diameter of about 90 A˚ (Fig 2B) The

interactions between the two monomers in the dimer are

extensive, involving the FAD binding site, whereas those

between the two dimers are mainly of helix–helix

inter-actions, similar to the ones seen in a four-helix bundle

structure The bound FAD has an extended conformation

with the isoalloxazine ring located at the crevice between the

two a-helix domains and the b-sheet domain within one

monomer, and its adenosine moiety lies at the interface

between two monomers (Fig 2) The fatty-acyl portion of

the thioester substrate is bound at the re-face of the flavin

ring buried inside the monomer between helices G and E

and the loop between J and K (Fig 2A) The cavity is deep

enough to accommodate substrate with an acyl chain length

of up to 12 carbons In addition, the base of the cavity is

wide and Ôupside down YÕ in shape so that it can

accommodate the x-end of the acyl chain of C12-CoA in

two different conformations, as seen in the structure of

human MCAD in complex with C12-CoA [5] Figure 3

shows the surface that lines the substrate-binding cavity of

MCAD The middle of the binding cavity is long and

narrow, just wide enough to accommodate the extended

pantetheine chain of the substrate On the other hand, the

adenosine-3¢-phosphate-5¢-diphosphate portion of the CoA

moiety is partially exposed to solvent at the interface of the

two monomers (Fig 3) This funnel shaped crevice

prob-ably serves as the entrance to the binding cavity The C2-C3

bond of the substrate is sandwiched between the carboxyl

group of Glu376 (catalytic base) and the isoalloxazine ring

of FAD, perfectly poised for the a-b dehydrogenation

reaction (Figs 3 and 4) The carbonyl oxygen of the

thioester substrate is hydrogen bonded to both the 2¢-OH

of the ribityl chain of FAD and the amide nitrogen of Glu376 [4] These interactions are important in the precise positioning and alignment of the flavin, substrate, and Glu376 for optimal catalysis In addition, they are respon-sible for the acidification of the a-proton of the substrate

No major changes in tertiary and quaternary structures have been observed upon binding the substrate; however, there are many subtle but significant changes in side chain conformation of the residues that line the active site cavity The most pronounced changes are observed in Glu376 (catalytic residue), Tyr375, and Glu99 The carboxylate of Glu376 moves toward to the C2 atom of the bound substrate poised to abstract the C2 proton The side chains

of Tyr375 and Glu99 adopt different conformations to accommodate the substrate In the absence of bound substrate, the active site cavity is occupied by a well-ordered string of water molecules that are successively displaced as the length of the fatty acyl chain increases until C12-CoA binds, at which point all the water molecules are expelled [19] This is probably how MCAD can accommodate substrates with a broad range of fatty acyl chain lengths with C8- and C10-CoA at the highest rate [9,20] For the shorter chain substrates (e.g C4-CoA or shorter), it must be entropically unfavorable to have several water molecules in the active site cavity, while the entrance of the cavity is blocked with the C4-CoA

Structures of other acyl-CoA dehydrogenases Four other ACAD structures have been determined: rat SCAD [3], a bacterial SCAD [21], human i3VD [6] and human GD [7] As expected from the primary sequence similarities, the overall polypeptide folds of these other ACADs are very similar to that of MCAD The root-mean-square deviations between Ca atoms of MCAD and those

of the other four ACAD structures that have been

Fig 3 A stereo view of the active site cavity of MCAD The cavity surface is shown with a green transparent surface The bound substrate, dodecanoyl-CoA, is shown with ball-and-sticks, with the last four carbon atoms (smaller balls) of the substrate modeled in two different conformations Residues lining the cavity are shown with stick models Atoms in the residues are colored as follows: carbon, black; nitrogen, blue; oxygen, red; sulfur, green; and phosphorus, purple The substrate is within the binding cavity, which is ÔoutsideÕ the molecular surface, and the lining residues are ÔinsideÕ the molecular surface The adenosine pyrophosphate moiety is partially exposed to solvent and the panthetheine-fatty acyl portion of the substrate is completely buried inside the molecule The molecular surface was generated with a 1.4 A˚ radius probe using the program [40].

determined to date range from 1.2 A˚ to 1.7 A˚, excluding the

N- and C-termini and the loop regions where a few

insertions and deletions occur (Fig 1) Furthermore, the

mode of substrate binding is also conserved, including the

two hydrogen bond interactions fromthe carbonyl of

the substrate thioester to the 2¢OH of the ribityl chain of the

FAD and to the main chain amide nitrogen of the residue

corresponding to Glu376 of MCAD Therefore, in this

section, only features unique to each of these other ACAD

structures will be discussed

Short chain acyl-CoA dehydrogenase

Structures of both rat SCAD (rSCAD) and a bacterial

SCAD from Megasphaera elsdenii (bSCAD) have been

determined [3,21] As expected, the catalytic residues are

Glu368 in the rat enzyme and Glu367 in the bacterial

enzyme, both of which are homologs of Glu376 in MCAD

(Fig 1) The binding cavities of rSCAD and bSCAD for the

fatty acyl moiety of the thioester substrate are shown in

Fig 5A and 5B, respectively, and they are very similar in

size and shape They are shallower than the cavity of

MCAD, which is consistent with the shorter chain substrate

specificity of SCADs Two features contribute to the

shallow binding cavities in SCADs: in SCADs, as well as

in i3VD and GD, all of which are specific for shorter

substrates than MCAD, there is one extra residue inserted in

the middle of helix E (Asn96 in rSCAD), compared to

helix E in MCAD (Fig 1) This insertion causes a bulge in

helix E and brings it closer to helix G, making the binding

cavity shallower In addition, there is a proline in the middle

of helix G in MCAD (Pro257 is conserved in all

mamma-lian MCADs; pig MCAD has an additional proline,

Pro258) This proline causes helix G to bend away from

the substrate, making its cavity deeper in comparison to

SCADs, i3VD and GD, which do not contain the proline It

is interesting to note that the insertion in helix E that makes

shallower cavities in SCAD, i3VD and GD is also missing in

the human LCAD sequence

Another interesting observation is that propionyl-CoA is

a poor substrate with bovine SCAD, though it is only one

methylene group shorter than the optimal substrate,

buty-ryl-CoA The kcatof propionyl-CoA with bovine SCAD is

0.1% of that with butyryl-CoA, and in fact propionyl-CoA

acts as a suicide inactivator by forming an adduct with FAD, having a partition ratio of 4.3 : 1 between productive catalysis and the suicide inhibition [22] The structural basis for this unexpected inhibition is not obvious at present Oxygen reactivity in the bacterial SCAD (bSCAD) bSCAD from M elsdenii reacts very rapidly with molecular oxygen compared to its mammalian counterpart [23] It should be noted that in M elsdenii, the enzyme physiolo-gically functions as an enoyl-CoA reductase rather than a dehydrogenase (i.e the reaction works in the opposite direction) and has no obvious need for protection against oxygen, since the organismis an anaerobe All known mammalian ACADs, except human iBD (which has a phenylalanine), have a tryptophan protecting the dimethyl-benzene side of the flavin ring at its si-face (Trp166 in MCAD) However, the bSCAD has a phenylalanine at the corresponding position, which makes its flavin more exposed to the solvent, which is consistent with the higher oxygen reactivity of the bacterial enzyme It would be interesting to see whether iBD also has a higher oxygen reactivity compared to other mammalian ACADs

The acyl-CoA binding cavity of iso(3)valeryl-CoA dehydrogenase (i3VD)

The structure of human i3VD reveals that, again, the overall polypeptide fold and its substrate-binding mode are very similar to that of MCAD [6] As in the case of SCAD, the acyl-CoA binding cavity is shallower than that of MCAD (see above), but it is also wider where the C3-C4 atoms of the substrate bind than the same region in either MCAD or SCAD This optimizes binding of the C3-branched, iso(3)valeryl moiety of the thioester substrate The specific activities of i3VD with butyryl-CoA and hexanoyl-CoA

as substrate are only 20% and 15% of the activity for isovaleryl-CoA, respectively [24] This C3-branched chain specificity comes from the absence of a bulky residue, tyrosine, at position 374 This tyrosine is conserved in all known acyl-CoA dehydrogenases except iBD, which has a leucine (also a bulky residue) at the corresponding position Figure 5C shows the binding cavity of i3VD with iso(3)val-eryl-CoA modeled into the site The lack of the tyrosine side

Fig 4 A stereo diagram showing comparison

of the catalytic base positions in MCAD and

i3VD The riboflavin portion of FAD (yellow)

and the acyl moiety of isovaleryl-CoA (purple)

are shown in ball-and-sticks The side chains

of Glu254 fromthe G helix of i3VD and

Glu376 fromthe loop between helices J and K

in MCAD are shown The red dotted lines

indicate proton abstraction fromthe C2 atom

of the substrate to the carboxylate of

gluta-mate and hydride transfer from the C3 atom

of the substrate to the N5 atomof FAD.

chain allows the C3-methyl group of the substrate to fit

snugly in the cavity provided by the absence of the phenol

ring This arrangement of the -C1-C2-C3- portion of the

iso(3)valeryl moiety is ideally suited to allow the pro-R C2

proton to be abstracted by the catalytic base, Glu254, and

hydride transfer fromC3 to the flavin On the other hand,

i2VD and iBD, both of which are specific for C2-branched

substrates, can tolerate a bulky residue (tyrosine in i2VD

and leucine in iBD) at this position However, the structural

basis for the C2-branched substrate specificity will require

the structure determination of i2VD, iBD, or both

Catalytic residue in long chain acyl- and iso(3)valeryl-CoA

dehydrogenase

Although chemical modification [25], crystallography [4]

and mutagenesis studies [26,27] firmly established Glu376

and its homologs as the catalytic residue in MCAD and

SCADs, this residue is not conserved in LCAD and i3VD

At the corresponding position, both rat and human LCAD

and rat i3VD have a glycine, whereas human i3VD has an

alanine Molecular modeling followed by site-specific

muta-genesis strongly suggests that Glu261 located in helix G of

LCAD is the catalytic residue [28] Crystal structure analysis

of human i3VD has confirmed that, indeed, Glu254 in i3VD (corresponding to Glu261 of LCAD) is the catalytic residue [6] Although Glu254 in i3VD is more than 100 residues away fromGlu376 in MCAD in the primary sequence (Fig 1), these two residues are topologically conserved in the three-dimensional structure (Fig 4) and carry out exactly the same chemistry of catalysis Furthermore, a double mutant of human MCAD, in which its catalytic base location has been changed to that of LCAD (Glu376Gly/ Thr255Glu), has been studied by biochemical and crystal-lographic methods [5,20] The resulting enzyme, medium long chain acyl-CoA dehydrogenase (MLCAD) has 20% of the activity of MCAD with octanoyl-CoA, and 25% of the activity of LCAD with dodecanoyl-CoA as the substrate This, together with the three-dimensional structure of MLCAD, provides further evidence that Glu255 can replace Glu376 as the catalytic residue in the structural frame of MCAD However, the structural basis for the observed change in the substrate chain length specificity, i.e a shift toward the longer chain substrates is not clear

at present A complete structural analysis of LCAD will probably shed light in this regard Similar mutational

Fig 5 Stereo views of the binding cavities

of (A) rat SCAD, (B) bacterial SCAD and (C) i3VD For clarity, only the binding cavities for the fatty-acyl moiety of the substrates are shown The surfaces of the ÔrimÕ of the cavities are very similar to that of MCAD shown in Fig 3 The orientation of each molecule is rotated approximately 120 about the y-axis fromthat shown in Fig 3 The cavities of both rSCAD (A) and bSCAD (B) are large enough

to comfortably accommodate hexanoyl-CoA – the last two carbon atoms shown with smaller, pale gray balls The ÔbaseÕ of the binding cavity

of i3VD is wider due to the lack of a tyrosine

at position 374, and the x-end of hexanoyl-CoA (smaller, pale gray balls) can bind to i3VD in two different conformations (C).

studies have also been carried out with i3VD [24], SCAD

[27], and i2VD [29], demonstrating that the catalytic

residues, Glu254 in i3VD, Glu368 in SCAD, and Glu381

in i2VD are homologs of Glu254 in LCAD and Glu376

in MCAD These results indicate that the catalytic residue in

ACADs can be placed either at 376 as in MCAD (or its

equivalent in other ACADs) or at 261 as in LCAD (or 254

in i3VD)

There are many families of distantly related enzymes in

which different functional groups are not conserved in the

primary sequences, but where catalytic atoms are in the

same position spatially [30] However, the family of ACADs

offers the only known example of a mechanistically

essentially identical series of enzymes in which the catalytic

residue is not conserved in the primary structures It also

serves as a reminder that any conclusions drawn from

sequence data alone should be taken with caution What

may have led to such a migration of the catalytic base in the

ACAD family? One possibility is that the ancestral gene had

glutamates at both locations and that the loss of either one

of themled to the evolution of ÔnewÕ, more efficient modern

enzymes A second possibility is that the ÔoldÕ, inefficient

enzyme had the catalytic residue at a distant position, and

the evolutionary optimization produced the ÔnewÕ, current

enzymes due to a functional group ÔhoppingÕ to either one of

the two current positions An example of a functional group

hopping can be found in the lipase family [31] A third

scenario involves gene (or exon) duplication, in which an

exon containing one glutamate duplicated itself at the other

location followed by mutations, resulting in migration of the

catalytic residue More complete comparisons of sequences

and structures are necessary for a better understanding of

the ACAD family lineage

Structure of glutaryl-CoA dehydrogenase (GD)

GD is unique among ACADs in that it catalyzes not only

the a-b dehydrogenation reaction but also decarboxylation

of the c-carboxylate of the substrate, glutaryl-CoA

Preli-minary results of structural studies of human GD have been

reported [7] The overall structure is the same as the other

known ACAD structures, and the identity of the catalytic

base, Glu370, is also confirmed The most striking difference

between the active site residues in GD and those of other

ACAD structures is the presence of a positively charged

residue, Arg94, at the base of the acyl moiety-binding site

Other ACADs have a neutral residue at this position A

model of glutaryl-CoA fitted into the active site suggests

that the c-carboxylate of the substrate would be within

hydrogen bonding distance fromthe guanidiniumgroup of

Arg94 Substitution of Arg94 to glycine or glutamine

increases the Kmfor glutaryl-CoA 10- to 16-fold compared

to the wild type enzyme, while the kcat of the mutant

enzymes decrease to 2–3% [32] These mutants are,

however, still capable of catalyzing the decarboxylation of

glutaconyl-CoA, suggesting that Arg94 is not absolutely

required for the decarboxylation reaction Thus, Arg94

appears to be involved in the binding of the substrate and in

the alignment of the glutaryl-CoA substrate for optimum

orientation for the dehydrogenation reaction In addition,

the positive charge of Arg94 appears to be involved in the

stabilization of the anionic intermediate, crotonyl-CoA

anion, during catalysis The exact mechanism and the structural basis for the decarboxylation reaction must await

a complete structural analysis of the enzyme in complex with substrate/analog

Electron transfer flavoprotein (ETF) and its interaction with ACADs

In mammalian mitochondria, the physiological electron acceptor of the ACADs is electron transfer flavoprotein (ETF), which is heterodimeric and contains one FAD and one AMP In addition to being the electron transfer partner

of the ACADs, ETF is also the physiological electron acceptor of two other flavoprotein dehydrogenases that are involved in choline metabolism (sarcosine dehydrogenase and dimethylglycine dehydrogenase) The structure of human ETF reveals that the molecule is comprised of three structural domains [33] Two domains are from the a-subunit and the third domain is composed entirely of the b-subunit The FAD lies at a cleft between the two subunits and is somewhat exposed to the solvent AMP is buried in the interior of the b-subunit and is not involved in the redox reaction, strongly suggesting that its role is purely structural Very little structural information is available regarding the interaction between mammalian ACADs and ETF Stable complex formation between these two flavoproteins has not been clearly demonstrated, although it has been reported recently that ETF forms soluble, relatively stable complexes with ACADs in mitochondria [34] As a result, to date, only

a hypothetical model of the complex of human ETF and porcine MCAD is available [33] In this model, electrons pass fromMCAD to ETF at the si-side of the MCAD flavin ring (Fig 6A), and the closest distance between the two flavins is about 19 A˚ (between the two dimethylbenzene rings) It is also possible that there are some conformational changes when the two molecules interact, allowing the two flavins to approach more closely for an efficient electron transfer More recent studies using small angle X-ray scattering techniques with human and Paracoccus ETF showed that the ETF molecules are indeed flexible enough to form multiple conformations in solution, strongly suggesting that ETF would adopt different conformations when it binds its electron transfer partners [35] However, detailed sites and the nature of interaction between the two electron transfer partners must await the structural analysis of a complex between ETF and one of the ACADs

Peroxisomal acyl-CoA oxidase Peroxisomal acyl-CoA oxidases (ACOs) are the peroxi-somal equivalent of the mitochondrial acyl-CoA dehydro-genases (ACADs) They are flavoenzymes containing one noncovalently bound FAD per subunit and belong to the same superfamily as ACADs [12] Like mitochondrial fatty acyl-CoA dehydrogenases, ACOs catalyze the initial and rate-determining step of the peroxisomal fatty acid b-oxidation pathway, i.e a,b-dehydrogenation of acyl-CoA, yielding trans-2-enoyl-CoA in the reductive half-reaction In the oxidative half-reaction of ACO, however, the reduced FAD is reoxidized by molecular oxygen producing hydrogen peroxide, whereas the reduced FAD

of ACADs transfer electrons to ETF, thus providing

electrons to the mitochondrial respiratory chain Compared

to the extensively studied ACADs, structural and

mechan-istic studies of ACOs have been relatively limited, mainly

due to the lack of their three-dimensional structure, which

has been obtained only very recently [8] The mechanisms of

the reductive half-reactions of ACO and ACAD are very

similar, but their physiological oxidative half-reactions are

completely different For example, reduced ACAD transfers

electrons to ETF one electron at a time, whereas ACO

transfers two electrons to molecular oxygen Therefore,

ACO and ACAD offer an excellent model system for

understanding how these two different oxidative half-reactions are controlled at the molecular level

Overall structure of rat ACO in comparison with MCAD

In rat peroxysomes, two acyl-CoA oxidase isozymes, ACO-I and ACO-II, have been identified, each of themhaving slightly different substrate acyl chain-length specificities [36]

As the structure of only ACO-II (optimal substrate acyl-chain length C14 compared to C10 for ACO-I) is known [8],

we hereafter refer to ACO-II as ACO for simplicity Figure 7A and 7B show the polypeptide fold of an ACO monomer and the overall structure of the dimeric molecule

of ACO, respectively Each subunit of ACO is comprised of four domains: N-terminal a-domain, N-terminal b-domain, C-terminal a-domain I, and C-terminal a-domain II (Fig 7A) The first three domains correspond to the entire subunit structure of acyl-CoA dehydrogenase, whereas the last domain of ACO, which is composed of the C-terminal

221 residues (C-terminal a-domain II), is not present in the ACAD structures (compare Fig 7A with 2A; Fig 1) The ACO dimer without the C-terminal a-domain II (Fig 7B minus the light brown and grey helices) and the MCAD dimer (Fig 7C) are very similar to each other However, a close inspection of the two reveals distinct features in each The relative orientation of the first two domains (the N-terminal a- and b-domains) with respect to the C-terminal a-domain I differs in ACO from the orientation

of the corresponding domains in MCAD by about 13, making the crevice between these domains wider, which in turn makes the binding cavity for the fatty acyl moiety wider and deeper (see the discussion of the shallower cavity in the SCAD structure, above) This 13 rotation also results in differences in the interaction between the flavin ring and the polypeptide chain of ACO, com pared to MCAD (e.g the number of hydrogen bonds between the flavin and the pro-tein m oiety is less in ACO than in MCAD) [8] The active-site cavity of ACO is 28 A˚ long and 6 A˚ wide and can accommodate the acyl-chain length of C23, in agreement with the acyl-chain length specificity previously determined [36] This wider active-site cavity in ACO is accessible not only to substrates with long acyl-chains in the reductive half-reaction, but also to molecular oxygen during the oxidative half-reaction

Structural basis for the regulation of oxygen reactivity

of ACO and ACADs, and of electron transfer to ETF

In order to understand the detailed molecular mechanism underlying the reactivity (or absence thereof) of reduced ACO and ACADs toward molecular oxygen or an electron acceptor protein, it is essential to consider the physical/ structural and chemical aspects associated with the flavin ring system The physical/structural aspect concerns how oxygen can physically access the reduced flavin embedded in the protein interior and how oxygen access is structurally ensured or prevented, whereas the chemical aspect concerns how electron transfer fromthe reduced flavin toward oxygen is chemically enhanced or impeded In the absence

of detailed knowledge of the electronic state of the reduced flavin in ACO with and without bound substrate/product, the following discussion of the oxidative half reaction of

Fig 6 Putative ETF docking site in MCAD and the corresponding site

in ACO (A) A view of MCAD indicating the hypothetical ETF

docking site as modeled by Roberts et al [33] a-Helical domain (red),

b-sheet domain (cyan), the first C-terminal a-domain (green) and the

FAD (yellow balls) The electrons are transferred fromthe MCAD

FAD to the ETF flavin through Trp166 and Met165 (B) The

cor-responding view of the ACO structure Helix S (grayish blue) of the

other subunit of the ACO dimer blocks the access of the ETF flavin to

the FAD of the ACO molecule.

ACO is limited only to the physical/structural aspect The

basic architecture of the active site cavity is remarkably

similar in ACO and MCAD (here used as the representative

of ACADs), reflecting their common mechanism for the

reductive half reaction The polypeptide segments surround-ing the flavin rsurround-ing and formsurround-ing the active site cavity in the two structures are nearly superimposable (rmsd of 0.46 A˚) [8], except for on the side of the pyrimidine moiety While the pyrimidine side of the flavin ring in MCAD is also covered by its polypeptide, the corresponding side in ACO

is exposed to solvent This difference results in reduced hydrogen bonding interactions between FAD and the polypeptide in the ACO structure compared to MCAD The solvent accessibility of the reduced flavin in ACO is probably responsible for its oxygen reactivity In contrast, the entire flavin ring in MCAD is well embedded in the protein interior, and thus oxygen access is physically restricted Only when the active site of MCAD is vacant, i.e when no ligand is bound, can the reduced flavin be exposed to molecular oxygen and solvent, resulting in oxygen reactivity, although not as high as that of a typical oxidase In the studies with the bacterial SCAD, the ratio of oxidase to dehydrogenase activity increases as the size of the CoA analog of the substrate decreases, which is consistent with the idea that the more solvent accessible the reduced flavin is, the higher the oxidase activity becomes [37] Another structural difference that further reinforces the difference in solvent accessibility can be seen in their quaternary structures The N-terminal side of the C-terminal a-domain II of ACO resides in the region corresponding to the interface between the two dimers of the MCAD tetramer, thus preventing the ACO dimers from associating with each other to forma tetramer Therefore, the active site of ACO is only partially protected by the small N-terminal side of the C-terminal a-domain II (Fig 7B), while the MCAD active site is more fully protected by a much bulkier subunit This again allows ACO easily accessible oxygen to its active site

Roberts et al [33] have postulated that, in the oxidative half-reaction of MCAD, ETF approaches MCAD (Fig 6A), forming an electron-transfer complex in which electrons are transferred fromthe si-face of the reduced flavin of MCAD to the oxidized flavin of ETF Figure 6B depicts the region of ACO corresponding to the proposed docking surface of MCAD to ETF It is noteworthy that helix S of ACO, the C-terminal end of the C-terminal a-domain II of the neighboring subunit in the dimer, covers the si-face of the flavin ring to be further away fromthe protein surface, thereby interfering with access of ETF to the surface of ACO Consequently, although electron acceptors corresponding to ETF are not known in peroxi-somes, ACO is protected from forming an electron-transfer complex with an ETF-like molecule, should one exist Therefore, the structural basis for ACO being an oxidase rather than a dehydrogenase is related to (a) oxygen accessibility to the active-site and the dimeric structure rather than a tetrameric form and (b) the si-face of the flavin ring in ACO being further away fromthe surface of the molecule than in MCAD due to the helix S, thereby preventing ACO fromforming an efficient electron transfer complex with an ETF-like molecule

Acknowledgements

This work was supported by National Institutes of Health Grant GM29076 (J.-J.P.K.) and Grants-in-Aid for Scientific Research on

Fig 7 Ribbon diagrams of the structure of ACO and comparison to

MCAD (A) A monomer of ACO The four domains of the ACO

monomer are shown: from N- to C-terminus, N-terminal a-domain

(red), b-domain (cyan), C-terminal a-domain I (green), and C-terminal

a-domain II (brown) The FAD is shown with yellow balls Note the

similarity between the ACO structure without C-terminal a-domain II

(brown) and that of the MCAD monomer shown in Fig 2A (B) A

dimer of ACO with the second subunit in blue (the N-terminal three

domains) and light grayish blue (C-terminal a-domain-II) (C) A dimer

of MCAD in a sim ilar orientation to that of ACO shown in (B) With

the exception of C-terminal a-domain II in ACO (shown in brown in

one subunit and its corresponding part of the second subunit in light

grayish blue), the two structures are very similar.

Priority Areas fromthe Ministry of Education, Culture, Sports, Science

and Technology of Japan (Category B: 13125206) (R.M.) The authors

thank Kevin Battaile for producing the figures.

References

1 Zhang, J., Zhang, W., Zou, D., Chen, G., Wan, T., Zhang, M &

Cao, X (2002) Cloning and functional characterization of

ACAD-9, a novel member of human acyl-CoA dehydrogenase

family Biochem Biophys Res Commun 297, 1033–1042.

2 Aoyama, T., Souri, M., Ushikubo, S., Kamijo, T., Yamaguchi, S.,

Kelley, R.I., Rhead, W.J., Uetake, K., Tanaka, K & Hashimoto,

T (1995) Purification of human very-long-chain acyl-coenzyme A

dehydrogenase and characterization of its deficiency in seven

patients J Clin Invest 95, 2465–2473.

3 Battaile, K.P., Molin-Case, J., Paschke, R., Wang, M., Bennett, D.,

Vockley, J & Kim, J.J (2002) Crystal structure of rat short chain

acyl-CoA dehydrogenase complexed with acetoacetyl-CoA:

com-parison with other acyl-CoA dehydrogenases J Biol Chem 277,

12200–12207.

4 Kim, J.J.P., Wang, M & Paschke, R (1993) Crystal structures of

medium-chain acyl-CoA dehydrogenase from pig liver

mitochondria with and without substrate Proc Natl Acad Sci.

USA 90, 7523–7527.

5 Lee, H.J., Wang, M., Paschke, R., Nandy, A., Ghisla, S & Kim,

J.J (1996) Crystal structures of the wild type and the Glu376Gly/

Thr255Glu mutant of human medium-chain acyl-CoA

dehydrogenase: influence of the location of the catalytic base on

substrate specificity Biochemistry 35, 12412–12420.

6 Tiffany, K.A., Roberts, D.L., Wang, M., Paschke, R., Mohsen,

A.W., Vockley, J & Kim, J.J (1997) Structure of human

iso-valeryl-CoA dehydrogenase at 2.6 A˚ resolution: structural basis

for substrate specificity Biochemistry 36, 8455–8464.

7 Kim, J.J.P., Wang, M & Paschke, R (1999) The crystal structure

of glutaryl-CoA dehydrogenase In Flavins and Flavoproteins

(Ghisla, S., Kroneck, P., Macheroux, P & Sund, H., eds), pp.

539–542 Scientific Publications, Berlin.

8 Nakajima, Y., Miyahara, I., Hirotsu, K., Nishina, Y., Shiga, K.,

Setoyama, C., Tamaoki, H & Miura, R (2002)

Three-dimen-sional structure of the flavoenzyme acyl-CoA oxidase-II from rat

liver, the peroxisomal counterpart of mitochondrial acyl-CoA

dehydrogenase J Biochem (Tokyo) 131, 365–374.

9 Beinert, H (1963) Acyl Coenzyme A Dehydrogenases In The

Enzymes, 2nd edn (Boyer, P.D., Lardy, H & Myrback, K., eds),

pp 447–466 Academic Press, New York.

10 Thorpe, C., Matthews, R.G & Williams, C.H Jr (1979)

Acyl-coenzyme A dehydrogenase from pig kidney Purification and

properties Biochemistry 18, 331–337.

11 Wang, R & Thorpe, C (1991) Reactivity of medium-chain

acyl-CoA dehydrogenase toward molecular oxygen Biochemistry 30,

7895–7901.

12 Matsubara, Y., Indo, Y., Naito, E., Ozasa, H., Glassberg, R.,

Vockley, J., Ikeda, Y., Kraus, J & Tanaka, K (1989) Molecular

cloning and nucleotide sequence of cDNAs encoding the

precursors of rat long chain coenzyme A, short chain

acyl-coenzyme A, and isovaleryl-acyl-coenzyme A dehydrogenases.

Sequence homology of four enzymes of the acyl-CoA

dehydrogenase family J Biol Chem 264, 16321–16331.

13 Goodman, S.I., Kratz, L.E., DiGiulio, K.A., Biery, B.J.,

Good-man, K.E., Isaya, G & FrerGood-man, F.E (1995) Cloning of

glutaryl-CoA dehydrogenase cDNA, and expression of wild type and

mutant enzymes in Escherichia coli Hum Mol Genet 4, 1493–

1498.

14 Pedersen, J.I., Eggertsen, G., Hellm an, U., Andersson, U &

Bjorkhem, I (1997) Molecular cloning and expression of cDNA

encoding 3a,7a,12a-trihydroxy-5b-cholestanoyl-CoA oxidase fromrabbit liver J Biol Chem 272, 18481–18489.

15 Zhang, Y.X., Denoya, C.D., Skinner, D.D., Fedechko, R.W., McArthur, H.A., Morgenstern, M.R., Davies, R.A., Lobo, S., Reynolds, K.A & Hutchinson, C.R (1999) Genes encoding acyl-CoA dehydrogenase (AcdH) homologues from Streptomyces coelicolor and Streptomyces avermitilis provide insights into the metabolism of small branched-chain fatty acids and macrolide antibiotic production Microbiology 145, 2323–2334.

16 Spector, M.P., DiRusso, C.C., Pallen, M.J., Garcia del Portillo, F., Dougan, G & Finlay, B.B (1999) The medium-/long-chain fatty acyl-CoA dehydrogenase (fadF) gene of Salmonella typhimurium is a phase 1 starvation-stress response (SSR) locus Microbiology 145, 15–31.

17 Krasko, A., Schroder, H.C., Hassanein, H.M., Batel, R., Muller, I.M & Muller, W.E (1998) Identification and expression of the SOS response, aidB-like, gene in the marine sponge Geodia cydo-nium: implication for the phylogenetic relationships of metazoan acyl-CoA dehydrogenases and acyl-CoA oxidases J Mol Evol.

47, 343–352.

18 Kim, J.J & Wu, J (1990) Structural studies of medium-chain acyl-CoA dehydrogenase frompig liver mitochondria Prog Clin Biol Res 321, 569–576.

19 Kim, J.J.P., Wang, M., Djordjevic, S., Paschke, R & Bennett, D.W (1993) Three-dimensional structures of Acyl-CoA dehy-drogenases: structural basis of substrate specificity In Flavins and Flavopoteins (Yagi, K., ed.), pp 273–282 Walter de Gruyter, New York.

20 Nandy, A., Kieweg, V., Krautle, F.G., Vock, P., Kuchler, B., Bross, P., Kim, J.J., Rasched, I & Ghisla, S (1996) Medium-long-chain chimeric human Acyl-CoA dehydrogenase: medium-Medium-long-chain enzyme with the active center base arrangement of long-chain Acyl-CoA dehydrogenase Biochemistry 35, 12402–12411.

21 Djordjevic, S., Pace, C.P., Stankovich, M.T & Kim, J.J (1995) Three-dimensional structure of butyryl-CoA dehydrogenase from Megasphaera elsdenii Biochemistry 34, 2163–2171.

22 Shaw, L & Engel, P.C (1985) The suicide inactivation of ox liver short-chain acyl-CoA dehydrogenase by propionyl-CoA For-mation of an FAD adduct Biochem J 230, 723–731.

23 Williamson, G & Engel, P.C (1984) Butyryl-CoA dehydrogenase from Megasphaera elsdenii Specificity of the catalytic reaction Biochem J 218, 521–529.

24 Mohsen, A.W & Vockley, J (1995) Identification of the active site catalytic residue in human isovaleryl-CoA dehydrogenase Biochemistry 34, 10146–10152.

25 Powell, P.J & Thorpe, C (1988) 2-octynoyl coenzyme A is a mechanism-based inhibitor of pig kidney medium-chain acyl coenzyme A dehydrogenase: isolation of the target peptide Biochemistry 27, 8022–8028.

26 Bross, P., Engst, S., Strauss, A.W., Kelly, D.P., Rasched, I.

& Ghisla, S (1990) Characterization of wild-type and an active site mutant of human medium chain acyl-CoA dehydro-genase after expression in Escherichia coli J Biol Chem 265, 7116–7119.

27 Battaile, K.P., Mohsen, A.W & Vockley, J (1996) Functional role

of the active site glutamate-368 in rat short chain acyl-CoA dehydrogenase Biochemistry 35, 15356–15363.

28 Djordjevic, S., Dong, Y., Paschke, R., Frerman, F.E., Strauss, A.W & Kim, J.J (1994) Identification of the catalytic base in long chain acyl-CoA dehydrogenase Biochemistry 33, 4258–4264.

29 Binzak, B., Willard, J & Vockley, J (1998) Identification of the catalytic residue of human short/branched chain acyl-CoA dehydrogenase by in vitro mutagenesis Biochim Biophys Acta

1382, 137–142.