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• W2057648766 abstract "Plants produce a unique peroxisomal short chain-specific acyl-CoA oxidase (ACX4) for β-oxidation of lipids. The short chain-specific oxidase has little resemblance to other peroxisomal acyl-CoA oxidases but has an ∼30% sequence identity to mitochondrial acyl-CoA dehydrogenases. Two biochemical features have been linked to structural properties by comparing the structures of short chain-specific Arabidopsis thaliana ACX4 with and without a substrate analogue bound in the active site to known acyl-CoA oxidases and dehydrogenase structures: (i) a solvent-accessible acyl binding pocket is not required for oxygen reactivity, and (ii) the oligomeric state plays a role in substrate pocket architecture but is not linked to oxygen reactivity. The structures indicate that the acyl-CoA oxidases may encapsulate the electrons for transfer to molecular oxygen by blocking the dehydrogenase substrate interaction site with structural extensions. A small binding pocket observed adjoining the flavin adenine dinucleotide N5 and C4a atoms could increase the number of productive encounters between flavin adenine dinucleotide and O2. Plants produce a unique peroxisomal short chain-specific acyl-CoA oxidase (ACX4) for β-oxidation of lipids. The short chain-specific oxidase has little resemblance to other peroxisomal acyl-CoA oxidases but has an ∼30% sequence identity to mitochondrial acyl-CoA dehydrogenases. Two biochemical features have been linked to structural properties by comparing the structures of short chain-specific Arabidopsis thaliana ACX4 with and without a substrate analogue bound in the active site to known acyl-CoA oxidases and dehydrogenase structures: (i) a solvent-accessible acyl binding pocket is not required for oxygen reactivity, and (ii) the oligomeric state plays a role in substrate pocket architecture but is not linked to oxygen reactivity. The structures indicate that the acyl-CoA oxidases may encapsulate the electrons for transfer to molecular oxygen by blocking the dehydrogenase substrate interaction site with structural extensions. A small binding pocket observed adjoining the flavin adenine dinucleotide N5 and C4a atoms could increase the number of productive encounters between flavin adenine dinucleotide and O2. The central pathway for fatty acid breakdown in higher plants is via peroxisomal β-oxidation. Fatty acids enter the cycle in the form of acyl-CoA thioesters, and during one round of β-oxidation, the acyl chain is shortened by a two-carbon unit and one acetyl-CoA molecule is produced. The first step in the peroxisomal β-oxidation cycle is the introduction of a double bond into the acyl-CoA substrate, resulting in the formation of 2-trans-enoyl-CoA. This reaction is a two-step reaction catalyzed by the family of acyl-CoA oxidases (ACXs) 2The abbreviations used are: ACX, acyl-CoA oxidase; ACX1, long chain-specific acyl-CoA oxidase; ACX4, short chain-specific acyl-CoA oxidase; CoA, coenzyme A; AcAcCoA, acetoacetyl-CoA; ACD, acyl-CoA dehydrogenase; FAD, flavin adenine dinucleotide; GCD, glutaryl-CoA dehydrogenase; MCAD, medium chain-specific acyl-CoA dehydrogenase; SCAD, short chain-specific acyl-CoA dehydrogenase; VLCAD, very long chain-specific acyl-CoA dehydrogenase; TLS, translation/libration/screw; e, elementary charge. requiring flavin adenine dinucleotide (FAD) as a cofactor. The cofactor gets reduced to FADH- in the first half-reaction concomitant with acyl-CoA oxidation. FADH- is reoxidized by molecular oxygen in the second step, thereby generating H2O2, an intracellular signaling molecule. Unlike plants, two parallel and distinct β-oxidation pathways exist in mammals, the peroxisomal β-oxidation pathway and a mitochondrial β-oxidation pathway. In mitochondrial β-oxidation, the first step is catalyzed by the acyl-CoA dehydrogenase family (ACD) (1Ghisla S. Thorpe C. Eur. J. Biochem. 2004; 271: 494-508Crossref PubMed Scopus (242) Google Scholar), which is related to ACXs. The FADH- in ACDs is not, however, reoxidized by molecular oxygen but rather by another flavoprotein, the electron transfer flavoprotein, which transfers electrons to the electron transport chain. As a result, the oxidation of fatty acids/amino acids and the generation of ATP molecules are linked in mitochondrial β-oxidation (2Hoard-Fruchey H.M. Goetzman E. Benson L. Naylor S. Vockley J. J. Biol. Chem. 2004; 279: 13786-13791Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). It is puzzling that natural selection has not forced plants to utilize the mitochondrial electron transport chain for general lipid oxidation despite the apparent ATP advantage of this pathway. This might reflect less stress on the ATP requirement and a higher demand for lipid turnover and excess oxygen management in plants. Six genes for ACX isozymes have been identified in Arabidopsis thaliana. Five encode for proteins of ∼75 kDa (3Eastmond P.J. Hooks M. Graham I.A. Biochem. Soc. Trans. 2000; 28: 755-757Crossref PubMed Scopus (22) Google Scholar, 4Adham A.R. Zolman B.K. Millius A. Bartel B. Plant J. 2005; 41: 859-874Crossref PubMed Scopus (86) Google Scholar, 5Graham I.A. Eastmond P.J. Prog. Lipid Res. 2002; 41: 156-181Crossref PubMed Scopus (189) Google Scholar). The proteins have different but overlapping substrate chain length specificity (3Eastmond P.J. Hooks M. Graham I.A. Biochem. Soc. Trans. 2000; 28: 755-757Crossref PubMed Scopus (22) Google Scholar) and one, ACX1, has been linked to the synthesis of the plant hormone jasmonate (6Cruz C.M. Martinez C. Buchala A. Metraux J.P. Leon J. Plant Physiol. 2004; 135: 85-94Crossref PubMed Scopus (119) Google Scholar, 7Li C. Schilmiller A.L. Liu G. Lee G.I. Jayanty S. Sageman C. Vrebalov J. Giovannoni J.J. Yagi K. Kobayashi Y. Howe G.A. Plant Cell. 2005; 17: 971-986Crossref PubMed Scopus (250) Google Scholar). The majority of the characterized enzymes in the ACX family form dimers in solution and have an extra C-terminal domain (3Eastmond P.J. Hooks M. Graham I.A. Biochem. Soc. Trans. 2000; 28: 755-757Crossref PubMed Scopus (22) Google Scholar, 8Hooks M.A. Kellas F. Graham I.A. Plant J. 1999; 20: 1-13Crossref PubMed Scopus (84) Google Scholar, 9Nakajima Y. Miyahara I. Hirotsu K. Nishina Y. Shiga K. Setoyama C. Tamaoki H. Miura R. J. Biochem. (Tokyo). 2002; 131: 365-374Crossref PubMed Scopus (54) Google Scholar, 10Pedersen L. Henriksen A. J. Mol. Biol. 2005; 345: 487-500Crossref PubMed Scopus (35) Google Scholar) that is not found in short, medium, and long chain-specific ACDs. The extra C-terminal domain resembles the C-terminal domain observed in very long chain-specific ACDs, which also form dimers (11Souri M. Aoyama T. Cox G.F. Hashimoto T. J. Biol. Chem. 1998; 273: 4227-4231Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 12Souri M. Aoyama T. Hoganson G. Hashimoto T. FEBS Lett. 1998; 426: 187-190Crossref PubMed Scopus (31) Google Scholar). The last A. thaliana ACX gene encodes for a short chain-specific enzyme (ACX4). It is a 50-kDa enzyme with little sequence identity to the other ACXs, yet it still catalyzes the same reoxidation step with the transfer of electrons from FADH- to molecular oxygen (13Hayashi H. De Bellis L. Ciurli A. Kondo M. Hayashi M. Nishimura M. J. Biol. Chem. 1999; 274: 12715-12721Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In mammals, the peroxisomal β-oxidation does not go to completion. Instead short chain fatty acids are transferred to the mitochondrial β-oxidation cycle. The peroxisomal short chain-specific ACX is unique to plants (14De Bellis L. Gonzali S. Alpi A. Hayashi H. Hayashi M. Nishimura M. Plant Physiol. 2000; 123: 327-334Crossref PubMed Scopus (17) Google Scholar) and seems to be an evolutionary link between the two alternative β-oxidation systems. The very long chain-specific ACD could have evolved in a similar way to the very long chain-specific ACD. Short chainspecific ACX shares the functional, homotetrameric state of short, medium, and long chain-specific ACDs (15Fu Z. Wang M. Paschke R. Rao K.S. Frerman F.E. Kim J.J. Biochemistry. 2004; 43: 9674-9684Crossref PubMed Scopus (70) Google Scholar) and has a higher sequence identity to mitochondrial dehydrogenases (∼30%) than to other peroxisomal oxidases (∼15%). Of the nine known members of the ACD family, plant short chain ACXs have the highest sequence identity to glutaryl-CoA dehydrogenases (GCD) (15Fu Z. Wang M. Paschke R. Rao K.S. Frerman F.E. Kim J.J. Biochemistry. 2004; 43: 9674-9684Crossref PubMed Scopus (70) Google Scholar). Bacterial GCDs, which have not been structurally characterized, are the GCDs with highest sequence identity to plant short chain ACXs (∼45%). We have determined the first structure of a peroxisomal short chain-specific ACX, A. thaliana ACX4, with and without a substrate analogue bound in the active site. The structure of ACX4, together with the previously determined structure of A. thaliana long chain-specific ACX, sheds new light on the role of the oligomerization state for substrate pocket architecture and the role of acyl binding pocket solvent accessibility. A mechanism is proposed by which the ACX enzymes encapsulate electrons for transfer to molecular oxygen. ACX4 Expression, Purification, and Crystallization—The A. thaliana gene encoding non-His-tagged ACX4 was amplified by PCR using a cDNA clone (GenBank™ accession No. T46525) obtained from the Arabidopsis Biological Resource Center as a template. The primers used were 5′-CTATCGCCATATGGCGGTGCTTTCATCT-3′ and 5′-GTCATCTCGAGTCAGAGACGGCTACGTGTACGCGGT-3′. The amplified DNA was inserted into the pET24 vector (Novagen) using NdeI and XhoI restriction sites. The resulting plasmid was transformed into E. coli BL21(DE3), and the cells were grown in LB medium containing 50 mg/liter kanamycin at 37 °C. Protein expression was induced at an A600 of 0.6-0.8 by the addition of isopropyl 1-thio-β-d-galactopyranoside to a final concentration of 0.5 mm. The cells were harvested by centrifugation 3 h after induction. The harvested cells were resuspended in 10 ml of BugBuster (Novagen) per liter of cell culture, with the addition of 0.5 ml of lysozyme (25 mg/ml; Sigma) per 10 ml of BugBuster. After cell lysis, benzonase (Sigma) was added at 1 μl/10 ml of BugBuster. The cell debris was removed by centrifugation at 20,000 × g for 60 min. The protein solution was dialyzed against 30% (w/v) polyethylene glycol 8000 to reduce the volume to ∼8 ml. The concentrated sample was applied onto a size exclusion column (Superdex75, GE Healthcare) using 20 mm HEPES buffer, pH 7.0, 100 mm NaCl, and 10 μm FAD as a running buffer. Fractions containing enzyme were pooled and applied directly onto an anion exchange column (MonoQ; Pharmacia Corporation) equilibrated with 20 mm HEPES buffer, pH 7.0, containing 100 mm NaCl and 10 μm FAD. Highly purified ACX4 (95%) was collected in the flow through. The protein was dialyzed against 20 mm HEPES buffer, pH 7.0, and 10 μm FAD and concentrated to 10 mg/ml in a Centriprep YM-10 filter unit (Millipore). After flash freezing in liquid nitrogen, the protein was stored at -20 °C. Crystals were obtained by the hanging drop vapor diffusion technique mixing equal volumes (2 μl) of ACX4 (10 mg/ml in 20 mm HEPES buffer, pH 7.0, 10 μm FAD) and precipitating solution (10-14% (w/v) polyethylene glycol 8000, 100 mm cacodylate, pH 6.5) with the addition of 0.5 μl of 20% (v/v) benzamidine-HCl and 0.5 μl of 10mm AcAcCoA and equilibrating against precipitating solution (1000 μl) at 22 °C. The crystals developed over 4 days. Cryoprotection was achieved by transfer of the crystals to 25% (v/v) glycerol by gradually increasing the concentration by 5%. Crystallization conditions for His-tagged ACX4 without AcAcCoA have been reported previously (16Pedersen L. Henriksen A. Acta Crystallogr. Sect. D. 2004; 60: 1125-1128Crossref PubMed Scopus (6) Google Scholar). The use of the non-His-tagged construct and inclusion of AcAcCoA in the crystallization conditions dramatically improved the reproducibility and quality of the ACX4 crystals. Data Collection and Structure Determination—Diffraction data from the ACX4·AcAcCoA complex were collected at the I711 beam line, MAX-lab, Lund University, Sweden. The crystal form was non-isomorphous with the crystal form observed with the His-tagged enzyme (16Pedersen L. Henriksen A. Acta Crystallogr. Sect. D. 2004; 60: 1125-1128Crossref PubMed Scopus (6) Google Scholar). ACX4·AcAcCoA crystals belong to space group P3221, a = b = 144.7 Å, c = 149.2 Å, contain four molecules/asymmetric unit and have a Matthew's coefficient of 2.4 Å3/Da and a solvent content of 47%. The best ACX4·AcAcCoA crystals diffracted to 2.7 Å, whereas the previously characterized crystals of His-tagged ACX4 diffracted to 3.9 Å. Diffraction data were indexed and integrated with MOS-FLM (17Leslie A. MOSFLM, MRC Laboratory of Molecular Biology. 1999; (Cambrigde, England)Google Scholar) and scaled with SCALA (18Kabsch W. J. Appl. Crystallogr. 1998; 21: 916-924Crossref Scopus (1680) Google Scholar). The data collection statistics are given in Table 1.TABLE 1Data collection and refinement statisticsACX4:AcAcCoAHis6-ACX4Data collectionBeam line1711, MAX-lab1711, MAX-labWavelength1.082 Å1.008 ÅSpace groupP3221P3221Unit cella = b = 144.7 Å, c = 149.2 Åa = b = 198.6 Å, c = 150.4 ÅT(K)100100Resolution (Å)20.0-2.70 (2.85-2.70)aNumbers in parentheses refer to the outer resolution shell.30.0-3.90 (4.10-3.90)aNumbers in parentheses refer to the outer resolution shell.Rmerge (%)13.2 (59.5)aNumbers in parentheses refer to the outer resolution shell.15.0 (32.0)aNumbers in parentheses refer to the outer resolution shell.Completeness99.6 (99.9)aNumbers in parentheses refer to the outer resolution shell.99.8 (99.9)aNumbers in parentheses refer to the outer resolution shell.Mean I/σI9.6 (1.7)aNumbers in parentheses refer to the outer resolution shell.7.4 (2.0)aNumbers in parentheses refer to the outer resolution shell.Redundancy2.911.6Wilson B (Å2)5725RefinementNo. of non-H protein atoms12,70419,422No. of non-H ligand atoms386312No. of water molecules1640RcrystbRcryst = ∑hkl||Fobs| - |Fcalc||/∑hkl|Fobs|.18.6 (30.8)aNumbers in parentheses refer to the outer resolution shell.24.7 (24.6)aNumbers in parentheses refer to the outer resolution shell.RfreecRfree = Rcryst but calculated using 5% of the reflections not included in the refinement procedure. The Rfree reflections were picked in shells (53).23.2 (36.7)aNumbers in parentheses refer to the outer resolution shell.25.1 (27.2)aNumbers in parentheses refer to the outer resolution shell.Root mean square deviation from idealitydAs defined by Ref. 54.Bond length (Å)0.0080.005Bond angles (°)1.1660.880Estimated overall coordinate error based on maximum likelihood (Å)eEstimated by REFMAC5 (20).0.20.6Residual temperature factorsProtein atoms (Å2)32.545.3Ligand atoms (A/B/C/D molecule, Å2)39.845.2Water molecules (Å2)30.4Occupancy of AcAcCoA (A/B/C/D subunit)0.5/1.0/0.75/1.0a Numbers in parentheses refer to the outer resolution shell.b Rcryst = ∑hkl||Fobs| - |Fcalc||/∑hkl|Fobs|.c Rfree = Rcryst but calculated using 5% of the reflections not included in the refinement procedure. The Rfree reflections were picked in shells (53Adams P.D. Pannu N.S. Read R.J. Brünger A.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5018-5023Crossref PubMed Scopus (383) Google Scholar).d As defined by Ref. 54Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2548) Google Scholar.e Estimated by REFMAC5 (20Collaborative Computing Project No. 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Open table in a new tab The structure of ACX4·AcAcCoA was solved by the molecular replacement technique as implemented in the program Phaser (19McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. Sect. D. 2005; 61: 458-464Crossref PubMed Scopus (1602) Google Scholar) included in the CCP4 program package (20Collaborative Computing Project No. 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) with a tetramer of rat short chain acyl-CoA dehydrogenase (21Battaile K.P. Molin-Case J. Paschke R. Wang M. Bennett D. Vockley J. Kim J.J. J. Biol. Chem. 2002; 277: 12200-12207Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) as the search template. The structure of the His-tagged ACX4 was solved by molecular replacement using MOLREP (22Vagin A.A. Isupov M.N. Acta Crystallogr. Sect. D. 2001; 57: 1451-1456Crossref PubMed Scopus (188) Google Scholar) using one subunit of the refined ACX4·AcAcCoA structure as model. The crystal had six molecules in the asymmetric unit. The ACX4·AcAcCoA structure was refined using iterative cycles of crystallography NMR software-simulated annealing refinement against the maximum likelihood target (23Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) or REFMAC5 (24Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) refinement alternating with manual rebuilding of the molecular structure using the O graphics software (25Jones A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). After resetting Biso values to 20, a last round of REFMAC5 refinement, including translation/libration/screw (TLS) refinement, was applied. This was followed by restrained positional refinement and supplemental Biso refinement (26Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D. 2001; 57: 122-133Crossref PubMed Scopus (1654) Google Scholar). Each of the four subunits defined a TLS group. Of the 439 residues in each subunit in the ACX4·AcAcCoA structure, all were comprised in the model, except 16 residues in the N terminus and 8 residues in the C terminus (including the PTS1 signal sequence). The occupancy of the AcAcCoA ligand was refined. Because of the low resolution of the His-tagged ACX4 data, only one round of crystallography NMR software-simulated annealing refinement was conducted, applying strict NCS between the six subunits in the asymmetric unit and not refining the B-factors nor including solvent molecules. The simulated annealing was followed by 10 cycles of TLS refinement in REFMAC5, first setting Biso = 20 for all protein atoms and applying one TLS group for each protein chain. Tight NCS restraints were used in the subsequent restrained positional refinement followed by refinement of the residual Biso. This procedure resulted in a drop in Rfree from 28 to 25%. In His-tagged ACX4, the His tag and N- and C-terminal residues were disordered. The final Rcryst and Rfree values of the ACX4·AcAcCoA structure were 19 and 23%, respectively, whereas the values for His-tagged ACX4 were 25 and 25%. The relatively low R-factor for His-tagged ACX4 at 3.9 Å resolution is probably a consequence of the use of the higher resolution ACX4·AcAcCoA structure as a starting point for the refinement. Refinement statistics are found in Table 1. Ramachandran plots of both structures have 92% of the residues in the most favored regions and one residue, Leu-393, in the γ-turn region. The coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with the accession numbers 2IX5 and 2IX6, respectively. Structural Analysis—Structural classification of the ACX4 structure took advantage of the SCOP data base (27Andreeva A. Howorth D. Brenner S.E. Hubbard T.J. Chothia C. Murzin A.G. Nucleic Acids Res. 2004; 32: D226-D229Crossref PubMed Google Scholar). The search for structural similarity to the N-terminal ACX4 extension was performed using the Macromolecular Structure data base secondary structure-matching facility against structures deposited in the PDB (28Krissinel E. Henrick K. Acta Crystallogr. Sect. D. 2004; 60: 2256-2268Crossref PubMed Scopus (3189) Google Scholar). Protein superposition was achieved with Lsqman (29Kleywegt G.J. Zou J.Y. Kjeldgaard M. Jones T.A. Rossmann M.A. Arnold E International Tables for Crystallography. F. Kluwer Academic Publishers, Dordrecht, The Netherlands2001Google Scholar), and protein-protein interactions were analyzed via the protein-protein interaction server (30Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20Crossref PubMed Scopus (2289) Google Scholar). Helanal (31Bansal M. Kumar S. Velavan R. J. Biomol. Struct. Dyn. 2000; 17: 811-819Crossref PubMed Scopus (149) Google Scholar), a program for characterization of helix geometry, was applied to analyze helix curvature, whereas secondary structure prediction for very long chain-specific acyl-CoA dehydrogenase (VLCAD) was done using Jpred (32Cuff J.A. Clamp M.E. Siddiqui A.S. Finlay M. Barton G.J. Bioinformatics. 1998; 14: 892-893Crossref PubMed Scopus (921) Google Scholar). The program Surfnet was used for binding pocket analysis (33Laskowski R.A. J. Mol. Graph. 1995; 13: 323-330Crossref PubMed Scopus (828) Google Scholar). In this analysis, a CoA molecule was left in the pocket to block the entrance, and the volume of the remaining cavity was calculated. Electrostatic potentials were calculated using the fullcharge force field and the program Delphi (Accelrys), setting the ionic strength of the solvent to 0.1 m and keeping default values for the other parameters. A similar result was obtained by calculating the electrostatic potentials using the Amber94 force field in the program MOE (Chemical Computing Group). Three-dimensional Structure—ACX4 was co-crystallized with the FAD cofactor and a substrate analogue, acetoacetyl-CoA (AcAcCoA). The ACX4 subunit adopted the canonical dehydrogenase fold with an N-terminal α-helical domain (residue 57-167), followed by a middle β-strand domain (residue 168-276) and a C-terminal four-helix bundle (residue 277-431) (Fig. 1A). The β-strand domain belongs to the acyl-CoA dehydrogenase NM domain-like superfamily and the C-terminal α-helical domain to the acyl-CoA dehydrogenase C-terminal domain-like superfamily. His-tagged and non-His-tagged A. thaliana ACX4, purified to homogeneity from E. coli (16Pedersen L. Henriksen A. Acta Crystallogr. Sect. D. 2004; 60: 1125-1128Crossref PubMed Scopus (6) Google Scholar), are both active as tetramers in solution (13Hayashi H. De Bellis L. Ciurli A. Kondo M. Hayashi M. Nishimura M. J. Biol. Chem. 1999; 274: 12715-12721Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 34Pedersen L. Structural Studies of Plant Acyl-CoA Oxidases, Ph.D. thesis. University of Copenhagen, Denmark2004Google Scholar). ACX4 also crystallizes as a tetramer with extensive intermolecular interactions between the subunits (Fig. 1C). The arrangement of the four molecules in the tetramer can be characterized as a dimer of dimers, where the dimer interface is formed mainly by interactions between the C-terminal α-domain of one subunit and the middle β-domain of the second subunit. Two FAD molecules are buried in each dimer interface (Fig. 1B). The four-helical bundle constituting the C-terminal α-helical domain forms the tetramer interface (Fig. 1C). The combined four-helix bundles of the dimer give the interface an almost flat shape. The extensive intermolecular interactions between the subunits in the crystal resemble the subunit arrangements characterized in mitochondrial dehydrogenase crystal structures (15Fu Z. Wang M. Paschke R. Rao K.S. Frerman F.E. Kim J.J. Biochemistry. 2004; 43: 9674-9684Crossref PubMed Scopus (70) Google Scholar, 21Battaile K.P. Molin-Case J. Paschke R. Wang M. Bennett D. Vockley J. Kim J.J. J. Biol. Chem. 2002; 277: 12200-12207Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 35Battaile K.P. Nguyen T.V. Vockley J. Kim J.J. J. Biol. Chem. 2004; 279: 16526-16534Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 36Djordjevic S. Pace C.P. Stankovich M.T. Kim J.J. Biochemistry. 1995; 34: 2163-2171Crossref PubMed Scopus (96) Google Scholar, 37Kim J.J. Wang M. Paschke R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7523-7527Crossref PubMed Scopus (267) Google Scholar, 38Lee H.J. Wang M. Paschke R. Nandy A. Ghisla S. Kim J.J. Biochemistry. 1996; 35: 12412-12420Crossref PubMed Scopus (70) Google Scholar, 39Tiffany K.A. Roberts D.L. Wang M. Paschke R. Mohsen A.W. Vockley J. Kim J.J. Biochemistry. 1997; 36: 8455-8464Crossref PubMed Scopus (94) Google Scholar) and define the biologically relevant homotetramer arrangement. A pair of cysteine residues is located on the dimer interface with a Cα-Cα distance of 8 Å (Fig. 1B). The cysteines do not form a disulfide bond in the crystal structure but could potentially do so in vivo. However, neither expression of ACX4 in the thioredoxin-deficient E. coli Origami2 strain (Novagen) nor the treatment of the reduced ACX4 with H2O2 results in disulfide bond formation judged by non-reduced SDS-PAGE analysis (results not shown). An N-terminal extension consisting of 56 amino acids is present in A. thaliana ACX4. The last 40 amino acid residues in the extension are well ordered and have been included in the model (Fig. 1, A and B). The well ordered part of the ACX4 N terminus extends toward the adjacent subunit, embracing the dimer (Fig. 1B). Also the C-terminal part of ACX4 is somewhat extended. The N- and C-terminal loops add to the stability of the ACX4 tetramer by expanding the intermolecular interactions. The ACX4 dimer interface buries 3493 Å2, whereas the short chainspecific acyl-CoA dehydrogenase (SCAD) interface is only 1751 Å2. The extensions account for 87% of this expansion of interface. Preventing Electrons from Going Astray—The N-terminal extension of ACX4 caps the region, which has been shown to be the site for electron transfer to the electron transfer flavoprotein in ACDs (40Toogood H.S. van Thiel A. Basran J. Sutcliffe M.J. Scrutton N.S. Leys D. J. Biol. Chem. 2004; 279: 32904-32912Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Removal of the tail opens a solvent-accessible channel to the FAD molecule analogous to the channel found in ACDs (Fig. 2). When superposing ACX1 and ACX4, the C terminus of ACX1 was found, surprisingly, to shield the corresponding area in the long chain-specificacyl-CoA oxidase (Fig.3). It is striking that, although ACX1 and ACX4 share only 17% sequence identity and 74% superposable Cα-atoms, the N-terminal extension of ACX4 superimposes almost perfectly on the C-terminal extension of ACX1. This strongly indicates that the N-terminal extension could have a potential role in electron transfer.FIGURE 3Superposition of A. thaliana ACX1 (green and gray) and ACX4 (beige and black) dimer. The N-terminal extension of ACX4 (red) covering the electron transfer flavoproteins docking area of ACDs is superposable on the C terminus of ACX1 (orange).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The extended C terminus of ACX4 also contributes to the shielding of the FAD cofactor. It bends back over the αG-αH loop of the C-terminal four-helix bundle to cover the adenine moiety of the FAD molecule (Fig. 2). The combined action of the two extensions completely buries the cofactor in ACX4. Blocking the access of substrate for the FAD cofactor reoxidation has been observed to be a feature used by other FAD-containing enzymes to favor oxidase activity over dehydrogenase activity. One example is the xanthine dehydrogenase/oxidase (41Enroth C. Eger B.T. Okamoto K. Nishino T. Nishino T. Pai E.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10723-10728Crossref PubMed Scopus (592) Google Scholar). N-terminal extensions of various lengths are also present in putative short chain acyl-CoA oxidase sequences from other plants, such as rice (UniProt Q5ZEL4), corn (Unigene Zm.8995), barley (Unigene Hv.567), soybean (Unigene Gma.6433), tomato (Unigene Les.2824), and potato (Unigene Stu.11425). Searching the PDB using the Macromolecular Structure data base secondary structure-matching facility (28Krissinel E. Henrick K. Acta Crystallogr. Sect. D. 2004; 60: 2256-2268Crossref PubMed Scopus (3189) Google Scholar) to identify and align similar proteins reveals that the N-terminal extension is not found in the structures of characterized ACDs. An N-terminal extension of 20-50 amino acids is present in the sequence of the mitochondrial ACDs, targeting the protein to the mitochondria; this peptide is, however, cleaved off upon import (42Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (981) Google Scholar). The human GCD (PDB accession code 1SIR) (15Fu Z. Wang M. Paschke R. Rao K.S. Frerman F.E. Kim J.J. Biochemistry. 2004; 43: 9674-9684Crossref PubMed Scopus (70) Google Scholar) has a high sequence identity to ACX4 (33%) as well as a high structural resemblance (root mean square deviation of 1.6 Å over 98% of the Cα atoms). Human GCD has a cleavable 44-amino-acidlong N-terminal mitochondrial targeting sequence with no significant homology to the N-terminal sequence of ACX4 (43Pearson W.R. Methods Enzymol. 1996; 266: 227-258Crossref PubMed Google Scholar). The N-terminal extension is found only in a few of the bacterial GCDs that share high sequence identity with ACX4. The Substrate Binding Pocket—The substrate analogue AcAcCoA is bound with the thioester bond sandwiched between the catalytic base and the FAD isoalloxazine ring in a cavity between the β-strand domain and the C-terminal four-helix bundle (Fig. 4). The ACX4·AcAcCoA c" @default.
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