Acyl-CoA oxidase 1 from Arabidopsis thaliana. Structure of a key enzyme in plant lipid metabolism

The peroxisomal acyl-CoA oxidase family plays an essential role in lipid metabolism by catalyzing the conversion of acyl-CoA into trans-2-enoyl-CoA during fatty acid beta-oxidation. Here, we report the X-ray structure of the FAD-containing Arabidopsis thaliana acyl-CoA oxidase 1 (ACX1), the first three-dimensional structure of a plant acyl-CoA oxidase. Like other acyl-CoA oxidases, the enzyme is a dimer and it has a fold resembling that of mammalian acyl-CoA oxidase. A comparative analysis including mammalian acyl-CoA oxidase and the related tetrameric mitochondrial acyl-CoA dehydrogenases reveals a substrate-binding architecture that explains the observed preference for long-chained, mono-unsaturated substrates in ACX1. Two anions are found at the ACX1 dimer interface and for the first time the presence of a disulfide bridge in a peroxisomal protein has been observed. The functional differences between the peroxisomal acyl-CoA oxidases and the mitochondrial acyl-CoA dehydrogenases are attributed to structural differences in the FAD environments.     

Gene isolation and characterization of two acyl CoA oxidases from soybean with broad substrate specificities and enhanced expression in the growing seedling axis

The first committed step in the -oxidation of fatty acids is catalyzed by the enzyme acyl-CoA oxidase (ACOX), which oxidizes a fatty acyl-CoA to a 2-trans-enoyl-CoA. To understand the role of -oxidation during seedling growth in soybean, two ACOX cDNAs were isolated by screening a seedling library with a DNA fragment obtained by RT-PCR by using degenerate oligonucleotides. The two cDNAs (ACX1;1 and ACX1;2) are 86% identical to each other at the nucleotide and the amino acid level. Their deduced amino acid sequences share significant homology with known acyl-CoA oxidases, including the conserved CGGHGY motif, a putative flavin mononucleotide binding site. In both sequences, the last three amino acids, ARL, represent a putative peroxisome targeting signal. The mRNA and protein of both cDNAs accumulated in all seedling tissues, with relatively stronger expression in the growing seedling axis and hypocotyl, and weaker expression in the cotyledon. Immunolocalization studies indicated that the two proteins were localized in the phloem cells of hypocotyl tissue. The two cDNAs were expressed in Escherichia coli and shown to possess acyl-CoA oxidase activity. With fatty acyl-CoA substrates of varying chain lengths, it was demonstrated that both ACX1;1 and ACX1;2 have broad substrate specificities (C₈–C₁₈). The stronger expression of ACX1;1 and ACX1;2 in the axis and hypocotyl tissue, the weaker expression in the oil-rich cotyledon tissue, and the broad substrate specificities suggest that the two acyl-CoA oxidases might play a general house-keeping role during soybean seedling growth, such as the turnover of membrane lipids.     

Acyl-CoA dehydrogenases and acyl-CoA oxidases. Structural basis for mechanistic similarities and differences.

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.     

Crystal structure of rat short chain acyl-CoA dehydrogenase complexed with acetoacetyl-CoA: comparison with other acyl-CoA dehydrogenases.

The acyl-CoA dehydrogenases are a family of flavin adenine dinucleotide-containing enzymes that catalyze the first step in the beta-oxidation of fatty acids and catabolism of some amino acids. They exhibit high sequence identity and yet are quite specific in their substrate binding. Short chain acyl-CoA dehydrogenase has maximal activity toward butyryl-CoA and negligible activity toward substrates longer than octanoyl-CoA. The crystal structure of rat short chain acyl-CoA dehydrogenase complexed with the inhibitor acetoacetyl-CoA has been determined at 2.25 A resolution. Short chain acyl-CoA dehydrogenase is a homotetramer with a subunit mass of 43 kDa and crystallizes in the space group P321 with a = 143.61 A and c = 77.46 A. There are two monomers in the asymmetric unit. The overall structure of short chain acyl-CoA dehydrogenase is very similar to those of medium chain acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, and bacterial short chain acyl-CoA dehydrogenase with a three-domain structure composed of N- and C-terminal alpha-helical domains separated by a beta-sheet domain. Comparison to other acyl-CoA dehydrogenases has provided additional insight into the basis of substrate specificity and the nature of the oxidase activity in this enzyme family. Ten reported pathogenic human mutations and two polymorphisms have been mapped onto the structure of short chain acyl-CoA dehydrogenase. None of the mutations directly affect the binding cavity or intersubunit interactions.     

The reductive half-reaction in acyl-CoA oxidase from Candida tropicalis: interaction with acyl-CoA analogues and an unusual thioesterase activity

A series of acyl-CoA analogues has been used to probe the substrate binding site and reductive half-reaction of acyl-CoA oxidase from the alkane utilizing yeast Candida tropicalis. Alkyl-SCoA thioethers, from octyl- to hexadecyl-SCoA, bind to the oxidase with progressively larger spectral perturbation of the flavin chromophore and with an incremental binding energy of about 260 cal/methylene group. The hydrocarbon binding subsite for acyl-CoA oxidase appears extensive and only weakly hydrophobic. CoA binding per se appears to contribute about 2.8 kcal to the observed binding energy. A number of acyl-CoA analogues such as 3-thia-acyl-, 3-oxa-acyl-, trans-3-enoyl-, and 3-keto-acyl-CoA derivatives form charge transfer complexes with the oxidase, but these long wavelength bands are both less pronounced and much less stable than those encountered with the acyl-CoA dehydrogenases. This instability reflects an intrinsic thioesterase activity of the oxidase which is observed with those ligands forming enolate to oxidized flavin charge-transfer complexes, but not with normal substrates such as palmitoyl-CoA. Chemical precedent suggests that these enzyme-bound enolates eliminate CoA via a ketene intermediate. The differences in behavior between acyl-CoA oxidase and dehydrogenase toward the ligands used in this work are discussed in terms of the need to exclude oxygen from productive encounters with substrate-reduced dehydrogenase.     

Reactivity of medium-chain acyl-CoA dehydrogenase toward molecular oxygen

The free two-electron-reduced form of medium-chain acyl-CoA dehydrogenase is reoxidized by 120 microM molecular oxygen (50 mM phosphate buffer, pH 7.6, 2 degrees C) with a half-time of approximately 7 s. Reoxidation yields hydrogen peroxide as a major product with only traces of the superoxide anion. In contrast, enzyme reduced with octanoyl-CoA is extremely slowly reoxidized oxygen, and so a series of 14 different substrate analogues have been tested to assess the structural factors responsible for this effect. Complexes with redox-inactive ligands such as 3-thia- and 2-azaoctanoyl-CoA lead to an approximately 3000-fold slowing of the rate of reoxidation of the free dihydroflavin form of the enzyme. Comparable ligands lacking the thioester carbonyl function are much less effective with rates some 1.3-4-fold slower than the free enzyme. The strong suppression of oxygen reactivity observed with certain ligands is probably not simply a steric effect but may reflect desolvation of the active site and consequent destabilization of the superoxide anion intermediate formed during reoxidation of the flavin. The profound differences in oxygen reactivity between acyl-CoA dehydrogenase and acyl-CoA oxidase and the unusual stability of certain flavoprotein semiquinones in air are discussed in terms of these thermodynamic and kinetic arguments.     

Three-dimensional structure of the flavoenzyme acyl-CoA oxidase-II from rat liver, the peroxisomal counterpart of mitochondrial acyl-CoA dehydrogenase

Acyl-CoA oxidase (ACO) catalyzes the first and rate-determining step of the peroxisomal beta-oxidation of fatty acids. The crystal structure of ACO-II, which is one of two forms of rat liver ACO (ACO-I and ACO-II), has been solved and refined to an R-factor of 20.6% at 2.2-A resolution. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into the N-terminal alpha-domain, beta-domain, and C-terminal alpha-domain. The X-ray analysis showed that the overall folding of ACO-II less C-terminal 221 residues is similar to that of medium-chain acyl-CoA dehydrogenase (MCAD). However, the N-terminal alpha- and beta-domains rotate by 13 with respect to the C-terminal alpha-domain compared with those in MCAD to give a long and large crevice that accommodates the cofactor FAD and the substrate acyl-CoA. FAD is bound to the crevice between the beta- and C-terminal domains with its adenosine diphosphate portion interacting extensively with the other subunit of the molecule. The flavin ring of FAD resides at the active site with its si-face attached to the beta-domain, and is surrounded by active-site residues in a mode similar to that found in MCAD. However, the residues have weak interactions with the flavin ring due to the loss of some of the important hydrogen bonds with the flavin ring found in MCAD. The catalytic residue Glu421 in the C-terminal alpha-domain seems to be too far away from the flavin ring to abstract the alpha-proton of the substrate acyl-CoA, suggesting that the C-terminal domain moves to close the active site upon substrate binding. The pyrimidine moiety of flavin is exposed to the solvent and can readily be attacked by molecular oxygen, while that in MCAD is protected from the solvent. The crevice for binding the fatty acyl chain is 28 A long and 6 A wide, large enough to accommodate the C23 acyl chain.     

A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes

Short-chain acyl-CoA oxidases are beta-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete beta-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform beta-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid beta-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid beta-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete beta-oxidation of acyl-CoA.     

Mutations in Arabidopsis acyl-CoA oxidase genes reveal distinct and overlapping roles in beta-oxidation

Indole-3-butyric acid (IBA) is an endogenous auxin used to enhance rooting during propagation. To better understand the role of IBA, we isolated Arabidopsis IBA-response (ibr) mutants that display enhanced root elongation on inhibitory IBA concentrations but maintain wild-type responses to indole-3-acetic acid, the principle active auxin. A subset of ibr mutants remains sensitive to the stimulatory effects of IBA on lateral root initiation. These mutants are not sucrose dependent during early seedling development, indicating that peroxisomal beta-oxidation of seed storage fatty acids is occurring. We used positional cloning to determine that one mutant is defective in ACX1 and two are defective in ACX3, two of the six Arabidopsis fatty acyl-CoA oxidase (ACX) genes. Characterization of T-DNA insertion mutants defective in the other ACX genes revealed reduced IBA responses in a third gene, ACX4. Activity assays demonstrated that mutants defective in ACX1, ACX3, or ACX4 have reduced fatty acyl-CoA oxidase activity on specific substrates. Moreover, acx1 acx2 double mutants display enhanced IBA resistance and are sucrose dependent during seedling development, whereas acx1 acx3 and acx1 acx5 double mutants display enhanced IBA resistance but remain sucrose independent. The inability of ACX1, ACX3, and ACX4 to fully compensate for one another in IBA-mediated root elongation inhibition and the ability of ACX2 and ACX5 to contribute to IBA response suggests that IBA-response defects in acx mutants may reflect indirect blocks in peroxisomal metabolism and IBA beta-oxidation, rather than direct enzymatic activity of ACX isozymes on IBA-CoA.     

Arabidopsis mutants in short- and medium-chain acyl-CoA oxidase activities accumulate acyl-CoAs and reveal that fatty acid beta-oxidation is essential for embryo development

The short-chain acyl-CoA oxidase (ACX4) is one of a family of ACX genes that together catalyze the first step of peroxisomal fatty acid beta-oxidation during early, postgerminative growth in oilseed species. Here we have isolated and characterized an Arabidopsis thaliana mutant containing a T-DNA insert in ACX4. In acx4 seedlings, short-chain acyl-CoA oxidase activity was reduced by greater than 98%, whereas medium-chain activity was unchanged from wild type levels. Despite the almost complete loss of short-chain activity, lipid catabolism and seedling growth and establishment were unaltered in the acx4 mutant. However, the acx4 seedlings accumulated high levels (31 mol %) of short-chain acyl-CoAs and showed resistance to 2,4-dichlorophenoxybutyric acid, which is converted to the herbicide and auxin analogue 2,4-dichlorophenoxyacetic acid by beta-oxidation. A mutant in medium-chain length acyl-CoA activity (acx3) (1) shows a similar phenotype to acx4, and we show here that acx3 seedlings accumulate medium-chain length acyl-CoAs (16.4 mol %). The acx3 and acx4 mutants were crossed together, and remarkably, the acx3acx4 double mutants aborted during the first phase of embryo development. We propose that acx3acx4 double mutants are nonviable because they have a complete block in short-chain acyl-CoA oxidase activity. This is the first demonstration of the effects of eliminating (short-chain) beta-oxidation capacity in plants and shows that a functional beta-oxidation cycle is essential in the early stages of embryo development.     

Structural analysis of the catalytic mechanism and stereoselectivity in Streptomyces coelicolor alditol oxidase

Alditol oxidase (AldO) from Streptomyces coelicolor A3(2) is a soluble monomeric flavin-dependent oxidase that performs selective oxidation of the terminal primary hydroxyl group of several alditols. Here, we report the crystal structure of the recombinant enzyme in its native state and in complex with both six-carbon (mannitol and sorbitol) and five-carbon substrates (xylitol). AldO shares the same folding topology of the members of the vanillyl-alcohol oxidase family of flavoenzymes and exhibits a covalently linked FAD which is located at the bottom of a funnel-shaped pocket that forms the active site. The high resolution of the three-dimensional structures highlights a well-defined hydrogen-bonding network that tightly constrains the substrate in the productive conformation for catalysis. Substrate binding occurs through a lock-and-key mechanism and does not induce conformational changes with respect to the ligand-free protein. A network of charged residues is proposed to favor catalysis through stabilization of the deprotonated form of the substrate. A His side chain acts as back door that "pushes" the substrate-reactive carbon atom toward the N5-C4a locus of the flavin. Analysis of the three-dimensional structure reveals possible pathways for diffusion of molecular oxygen and a small cavity on the re side of the flavin that may host oxygen during FAD reoxidation. These features combined with the tight shape of the catalytic site provide insights into the mechanism of AldO-mediated regioselective oxidation reactions and its substrate specificity.     

Chemical and catalytic properties of the peroxisomal acyl-coenzyme A oxidase from Candida tropicalis

The peroxisomal acyl-CoA oxidase has been purified from extracts of the yeast Candida tropicalis grown with alkanes as the principal energy source. The enzyme has a molecular weight of 552,000 and a subunit molecular weight of 72,100. Using an experimentally determined molar extinction coefficient for the enzyme-bound flavin, a minimum molecular weight of 146,700 was determined. Based on these data, the oxidase contains eight perhaps identical subunits and four equivalents of FAD. No other β-oxidation enzyme activities are detected in purified preparations of the oxidase. The oxidase flavin does not react with sulfite to form an N(5) flavin-sulfite complex. Photochemical reduction of the oxidase flavin yields a red semiquinone; however, the yield of semiquinone is strongly pH dependent. The yield of semiquinone is significantly reduced below pH 7.5. The flavin semiquinone can be further reduced to the hydroquinone. The behavior of the oxidase flavin during photoreduction and its reactivity toward sulfite are interpreted to reflect the interaction in the N(1)-C(2)O region of the flavin with a group on the protein which acts as a hydrogen-bond acceptor. Like the acyl-CoA dehydrogenases which catalyze the same transformation of acyl-CoA substrates, the oxidase is inactivated by the acetylenic substrate analog, 3-octynoyl-CoA, which acts as an active site-directed inhibitor.     

Crystal structure analysis of recombinant rat kidney long chain hydroxy acid oxidase

Long chain hydroxy acid oxidase (LCHAO) is a member of an FMN-dependent enzyme family that oxidizes L-2-hydroxy acids to ketoacids. LCHAO is a peroxisomal enzyme, and the identity of its physiological substrate is unclear. Mandelate is the most efficient substrate known and is commonly used in the test tube. LCHAO differs from most family members in that one of the otherwise invariant active site residues is a phenylalanine (Phe23) instead of a tyrosine. We now report the crystal structure of LCHAO. It shows the same beta8alpha8 TIM barrel structure as other structurally characterized family members, e.g., spinach glycolate oxidase (GOX) and the electron transferases yeast flavocytochrome b2 (FCB2) and Pseudomonas putida mandelate dehydrogenase (MDH). Loop 4, which is mobile in other family members, is visible in part. An acetate ion is present in the active site. The flavin interacts with the protein in the same way as in the electron transferases, and not as in GOX, an unexpected observation. An interpretation is proposed to explain this difference between GOX on one hand and FCB2 and LCHAO on the other hand, which had been proposed to arise from the differences between family members in their reactivity with oxygen. A comparison of models of the substrate bound to various published structures suggests that the very different reactivity with mandelate of LCHAO, GOX, FCB2, and MDH cannot be rationalized by a hydride transfer mechanism.     

Probing oxygen activation sites in two flavoprotein oxidases using chloride as an oxygen surrogate

A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX·chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX·chloride complex and a ternary MSOX·chloride·MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.     

Structure and function of plant acyl-CoA oxidases.

Acyl-CoA oxidases (in peroxisomes) and acyl-CoA dehydrogenases (in mitochondria) catalyse the first step in fatty acid beta-oxidation, the pathway responsible for lipid catabolism and plant hormone biosynthesis. The interplay and differences between peroxisomal and mitochondrial beta-oxidation processes are highlighted by the variation in the enzymes involved. Structure and sequence comparisons are made with a focus on the enzyme's mechanistic means to control electron transfer paths, reactivity towards molecular oxygen, and spatial and architectural requirements for substrate discrimination.     

α/β Barrel evolution and the modular assembly of enzymes: Emerging trends in the flavin oxidase/dehydrogenase family

α/β barrels have an ill-defined origin. Evidence exists which favours their divergent evolution from a common ancestral barrel and convergent evolution to a stable fold. However, recent sequence and structural information for the flavin oxidase/dehydrogenase family of barrel enzymes indicate that sub-families of α/β barrels have evolved divergently. The modular fusion of barrel domains with core structures from other gene families has also contributed to the evolution of related but catalytically distinct enzyme molecules within each sub-family of the flavin oxidases/dehydrogenases. An analysis of the structures and sequences of the flavin oxidases/dehydrogenases has now enabled studies focusing on the evolutionary origins and modular assembly of this important family of proteins to be initiated.     

Structures of Michaelis and product complexes of plant cytokinin dehydrogenase: implications for flavoenzyme catalysis

Cytokinins form a diverse class of compounds that are essential for plant growth. Cytokinin dehydrogenase has a major role in the control of the levels of these plant hormones by catalysing their irreversible oxidation. The crystal structure of Zea mays cytokinin dehydrogenase displays the same two-domain topology of the flavoenzymes of the vanillyl-alcohol oxidase family but its active site cannot be related to that of any other family member. The X-ray analysis reveals a bipartite architecture of the catalytic centre, which consists of a funnel-shaped region on the protein surface and an internal cavity lined by the flavin ring. A pore with diameter of about 4A connects the two active-site regions. Snapshots of two critical steps along the reaction cycle were obtained through the structural analysis of the complexes with a slowly reacting substrate and the reaction product, which correspond to the states immediately before (Michaelis complex) and after (product complex) oxidation has taken place. The substrate displays a "plug-into-socket" binding mode that seals the catalytic site and precisely positions the carbon atom undergoing oxidation in close contact with the reactive locus of the flavin. A polarising H-bond between the substrate amine group and an Asp-Glu pair may facilitate oxidation. Substrate to product conversion results in small atomic movements, which lead to a planar conformation of the reaction product allowing double-bond conjugation. These features in the mechanism of amine recognition and oxidation differ from those observed in other flavin-dependent amine oxidases.     

Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair

Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue. The x-ray structure of the enzyme from Brevibacterium sterolicum containing covalently bound FAD has been determined and refined to 1.7-A resolution. The active site consists of a cavity sealed off from the exterior of the protein. A model for the steroid substrate, cholesterol, can be positioned in the pocket revealing the structural factors that result in different substrate binding affinities between the two known forms of the enzyme. The structure suggests that Glu(475), located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic reaction. A water-filled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry point for molecular oxygen for the oxidative half-reaction. An arginine and a glutamate residue at the active site, found in two conformations are proposed to control oxygen access to the cavity from the channel. These concerted side chain movements provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the enzyme.     

High resolution structures of an oxidized and reduced flavoprotein. The water switch in a soluble form of (S)-mandelate dehydrogenase

The crystal structures of a soluble mutant of the flavoenzyme mandelate dehydrogenase (MDH) from Pseudomonas putida and of the substrate-reduced enzyme have been analyzed at 1.35-A resolution. The mutant (MDH-GOX2) is a fully active chimeric enzyme in which residues 177-215 of the membrane-bound MDH are replaced by residues 176-195 of glycolate oxidase from spinach. Both structures permit full tracing of the polypeptide backbone chain from residues 4-356, including a 4-residue segment that was disordered in an earlier study of the oxidized protein at 2.15 A resolution. The structures of MDH-GOX2 in the oxidized and reduced states are virtually identical with only a slight increase in the bending angle of the flavin ring upon reduction. The only other structural changes within the protein interior are a 10 degrees rotation of an active site tyrosine side chain, the loss of an active site water, and a significant movement of six other water molecules in the active site by 0.45 to 0.78 A. Consistent with solution studies, there is no apparent binding of either the substrate, mandelate, or the oxidation product, benzoylformate, to the reduced enzyme. The observed structural changes upon enzyme reduction have been interpreted as a rearrangement of the hydrogen bonding pattern within the active site that results from binding of a proton to the N-5 position of the anionic hydroquinone form of the reduced flavin prosthetic group. Implications for the low oxidase activity of the reduced enzyme are also discussed.     

Three-dimensional structures of glycolate oxidase with bound active-site inhibitors.

A key step in plant photorespiration, the oxidation of glycolate to glyoxylate, is carried out by the peroxisomal flavoprotein glycolate oxidase (EC 1.1.3.15). The three-dimensional structure of this alpha/beta barrel protein has been refined to 2 A resolution (Lindqvist Y. 1989. J Mol Biol 209:151-166). FMN dependent glycolate oxidase is a member of the family of alpha-hydroxy acid oxidases. Here we describe the crystallization and structure determination of two inhibitor complexes of the enzyme, TKP (3-Decyl-2,5-dioxo-4-hydroxy-3-pyrroline) and TACA (4-Carboxy-5-(1-pentyl)hexylsulfanyl-1,2,3-triazole). The structure of the TACA complex has been refined to 2.6 A resolution and the TKP complex, solved with molecular replacement, to 2.2 A resolution. The Rfree for the TACA and TKP complexes are 24.2 and 25.1%, respectively. The overall structures are very similar to the unliganded holoenzyme, but a closer examination of the active site reveals differences in the positioning of the flavin isoalloxazine ring and a displaced flexible loop in the TKP complex. The two inhibitors differ in binding mode and hydrophobic interactions, and these differences are reflected by the very different Ki values for the inhibitors, 16 nM for TACA and 4.8 microM for TKP. Implications of the structures of these enzyme-inhibitor complexes for the model for substrate binding and catalysis proposed from the holo-enzyme structure are discussed.