Spinach glycolate oxidase and yeast flavocytochrome b2 are structurally homologous and evolutionarily related enzymes with distinctly different function and flavin mononucleotide binding

A comparison of the three-dimensional structures of the flavin mononucleotide (FMN)-dependent enzymes glycolate oxidase, flavocytochrome b2, and trimethylamine dehydrogenase is presented. Their flavin-binding domains all have the same structural motif, the 8-fold beta/alpha-barrel domain, which is also present in a large number of other enzymes. FMN is bound in a similar fashion in all three enzymes. The binding site is at the carboxyl-terminal end of the eight beta-strands of the barrel where the active site is invariably found in this type of domain structure. The similarity of the structures of glycolate oxidase and flavocytochrome b2 extends to the loop regions and even outside the beta/alpha-barrels with a root mean square deviation of 0.93 A for 311 superimposed C alpha-atoms and with a sequence identity of 37%. A detailed analysis of their active sites shows, however, that the orientation of FMN is significantly different in the two structures due to different conformations of residues in the end of strand one. Thus, in flavocytochrome b2 a hydrogen bond is formed between the FMN N-5 position and the main chain amide of Ala-198, while in glycolate oxidase, the ring system is tilted away from the strand, creating a pocket on the re-side of the FMN ring where a water molecule is bound. Model building shows that this site could accommodate the hydroperoxide moiety of a FMN-4a-hydroperoxide intermediate. Thus, in the course of evolution, a few mutations in, and close to, the active sites have fine tuned these enzymes to exert their specific functions as an oxidase or transferase, respectively.     

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.     

Extreme pKa displacements at the active sites of FMN-dependent alpha-hydroxy acid-oxidizing enzymes.

Flavocytochrome b2 (or L-lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related alpha-hydroxy acid-oxidizing FMN-dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate alpha-proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three-dimensional structure of the enzyme leads to the conclusion that the high pKa belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free. The features of the three-dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the alpha-hydroxy acid-oxidizing flavooxidases, which are homologous to flavocytochrome b2. The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.     

Similarity of different β-strands flanked in loops by glycines and prolines from distinct (α/β)8-barrel enzymes: Chance or a homology?

Many (alpha/beta)8-barrel enzymes contain their conserved sequence regions at or around the beta-strand segments that are often preceded and succeeded by glycines and prolines, respectively. alpha-Amylase is one of these enzymes. Its sequences exhibit a very low degree of similarity, but strong conservation is seen around its beta-strands. These conserved regions were used in the search for similarities with beta-strands of other (alpha/beta)8-barrel enzymes. The analysis revealed an interesting similarity between the segment around the beta 2-strand of alpha-amylase and the one around the beta 4-strand of glycolate oxidase that are flanked in loops by glycines and prolines. The similarity can be further extended on other members of the alpha-amylase and glycolate oxidase subfamilies, i.e., cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, and flavocytochrome b2, respectively. Moreover, the alpha-subunit of tryptophan synthase, the (alpha/beta)8-barrel enzyme belonging to the other subfamily of (alpha/beta)8-barrels, has both investigated strands, beta 2 and beta 4, similar to beta 2 of alpha-amylase and beta 4 of glycolate oxidase. The possibilities of whether this similarity exists only by chance or is a consequence of some processes during the evolution of (alpha/beta)8-barrel proteins are briefly discussed.     

Carbanion versus hydride transfer mechanisms in flavoprotein-catalyzed dehydrogenations

The present understanding of the mechanisms by which flavoproteins oxidize amino acid or hydroxy acids to the respective imino or keto acids is reviewed. The observation that many of these enzymes catalyze the elimination of HBr or HCl from the appropriate beta-halogenated substrate was long considered evidence for a carbanion intermediate. Recent structural and mechanistic studies are not compatible with the intermediacy of carbanions in the reactions catalyzed by d-amino acid oxidase and flavocytochrome b(2). In contrast, the data are most consistent with mechanisms involving direct hydride transfer.     

High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent l-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b₂ (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC₅₀ in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 Å resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the ∼100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.     

Kinetic study of the activation of the neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and phenylarsine oxide

The O(2)(-) generating NADPH oxidase complex of neutrophils comprises two sets of components, namely a membrane-bound heterodimeric flavocytochrome b which contains the redox centers of the oxidase and water-soluble proteins of cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils in the presence of GTPgammaS and arachidonic acid. NADPH oxidase activation is inhibited by phenylarsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol groups. The site of action of PAO was localized by photolabeling in the beta-subunit of flavocytochrome b [Doussière, J., Poinas, A, Blais, C., and Vignais, P. V. (1998) Eur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocytochrome b [Doussière, J., Gaillard, J., and Vignais, P. V. (1996) Biochemistry 35, 13400-13410]. Here we report that the promoting effect of arachidonic acid on the activation of NADPH oxidase is due to specific binding of arachidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of flavocytochrome b for O(2), an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affected by arachidonic acid. We further demonstrate that PAO antagonizes the effect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O(2), but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flavocytochrome b at two different interacting sites.     

L-lactate 2-monooxygenase from Mycobacterium smegmatis. Cloning, nucleotide sequence, and primary structure homology within an enzyme family

L-Lactate 2-monooxygenase catalyzes the oxidation of L-lactate to acetate and carbon dioxide. The catalytic mechanism has been extensively investigated but very little is known about which amino acid residues may play a role in catalysis. As a first step toward this goal, the gene for this protein from Mycobacterium smegmatis has been cloned and sequenced. Peptide sequencing data for L-lactate 2-monooxygenase was used to construct three sets of fully redundant tetradecamer oligonucleotide probes, which were hybridized to restriction-digested M. smegmatis DNA. An approximately 3-kilobase pair PstI fragment hybridized with two of the probes. This region was subsequently isolated and cloned into Escherichia coli. From this size-fractionated gene bank, a 3.1-kilobase pair genomic DNA fragment was isolated by colony hybridization to two of the oligonucleotide probes. The complete gene for L-lactate 2-monooxygenase was contained on this fragment as shown by DNA sequencing of the whole insert. The DNA sequence codes for a mature protein that is 393 amino acids in length with a subunit molecular weight of 43,072 (including the FMN). The protein sequence shows impressive homology with the primary structures of two mechanistically related proteins, yeast flavocytochrome b2 (Lederer, F., Cortial, S., Becam, A.-M., Haumont, P.-Y., and Perez, L. (1985) Eur. J. Biochem. 152, 419-428; Guiard, B. (1985) EMBO J. 4, 3265-3272) and spinach glycolate oxidase (Volkita, M., and Somerville, C. R. (1987) J. Biol. Chem. 262, 15825-15828; Cederlund, E., Lindqvist, Y., Soderlund, G., Br?ndén, C.-I., and Jornvall, H. (1988) Eur. J. Biochem. 173, 523-530). For each residue proposed from the crystal structure of glycolate oxidase to be involved in catalysis (Lindqvist, Y., and Br?ndén, C.-I. (1989) J. Biol. Chem. 264, 3624-3628), an identical residue was found in a homologous position in lactate oxidase. Furthermore, most of these residues occur in regions whose sequences are highly conserved between lactate oxidase, flavocytochrome b2, and glycolate oxidase.     

Primary structure of glycolate oxidase from spinach

The primary structure of glycolate oxidase from spinach has been determined. Six different types of peptide digest were investigated, utilizing CNBr, proteolytic enzymes, and chemical modifications to change a specificity of cleavage. In total, 90 peptides were purified and analyzed. The studies were aimed at correlation with crystallographic analysis of the same protein carried through in parallel and with cDNA studies which utilized initially determined amino acid sequences for synthesis of oligonucleotide probes. Continuous comparisons with the results from the crystallographic studies helped at an early stage to secure peptide overlaps, at the same time as the peptide data secured residue assignments in the electron density maps. In the end, all data agree and regions from all parts of the molecule have been checked by independent methods of analysis. The primary structure establishes the type of N-terminal post-translational processing, and yields information on segments not fully defined in electron density maps. Combined, the chemical, crystallographic, and cDNA data give extensive reliability. The peptide analysis shows that the N-terminus is blocked by acylation of the initiator methionine, which is in a primary structure typical for non-removal of the methionine in the processing events of the nascent protein chain. The molecule is comparatively rich in menthionine and some other generally less common residues, but has only one cysteine residue and no extensive hydrophobic segment. An amino acid sequence homology with flavocytochrome b2 from yeast, as expected from known similarities in tertiary structure, is observed (33% residue identities).     

Hydroxamates as substrates and inhibitors for FMN-dependent 2-hydroxy acid dehydrogenases

Long-chain hydroxy acid oxydase (HAO) is a member of a flavoenzyme family with significant amino acid sequence similarity and strongly conserved three-dimensional structure; in particular, active-site amino acids involved in catalysis are invariant, with one exception, and numerous enzymatic studies suggest an identical chemical mechanism involving an intermediate carbanion for all family members. Known physiological substrates are a variety of L-2-hydroxy acids. Peroxisomal HAO differs from the other family members in that its actual physiological substrate is not known; it was first described as an L-amino acid oxidase, and recently was identified as an enzyme that converts creatol (hydroxycreatinine) to methylguanidine (a metabolite involved in a variety of uremic syndromes). Creatol (2-amino-5-hydroxy-1-methyl-4(5H)imidazolone) is not a 2-hydroxy acid. We show in this work that 2-hydroxyphenyl acetohydroxamate (HYPAH, the hydroxamate of mandelic acid), a compound that bears similarity both to mandelate (one of the best substrates known) and to creatol, is turned over by HAO, but between 10- and 100-fold less efficiently than mandelate itself. The compound also binds to the active site of homologous flavocytochrome b(2) (L-lactate dehydrogenase). Comparative pH-rate studies for mandelate and its hydroxamate suggest that HYPAH may bind in its ionized form. Both pH-rate profiles are bell-shaped curves, as are those determined for two other family members, flavocytochrome b(2) and mandelate dehydrogenase; while the group with an acid pK(a) between 5 and 6 is most likely the active-site histidine (the residue which abstracts the substrate C2 proton), the identity of the basic group is less clear. It has been proposed to be one of the active site arginines (Lehoux, I., and Mitra, B. (1999) Biochemistry38, 5836-5848); we suggest as an alternative that it could be the lysine residue that interacts with the flavin N1 and O2 positions and stabilizes the negative charge of reduced flavin. In addition to these studies, we have found that HAO is competitively inhibited by benzohydroxamate, which is one atom shorter than HYPAH; its affinity is nearly 100-fold lower than that of the substrate, in contrast to the strong inhibition it exerts on mandelate racemase (Maurice, St. M., and Bearne, S. L. (2000) Biochemistry39, 13324-13335). In the latter case, the 100-fold higher affinity compared to mandelate was proposed to arise from the fact that the hydroxamate can mimic the enolic intermediate which lies on the reaction pathway after C2 proton abstraction. Thus our results do not support the existence of a similar enolic intermediate for HAO (and probably its homologues), although they do not disprove it.     

Isolation and characterization of glycolic acid oxidase from human liver.

Glycolic acid oxidase has been isolated from human liver and purified over 3000-fold to a specific activity of 123 U/mg protein by a 5-step procedure. The preparation gave a single protein band on polyacrylamide gel electrophoresis, required flavin mononucleotide for catalytic activity, had a pH optimum between 8.2-8.8 depending on the substrate, and had a molecular weight of 105 000. The enzyme has a broad specificity towards alpha-hydroxy acids. Glycolate (Km = 3.3 . 10(-4) M) was the most effective substrate. The enzyme was stable for several months when stored as an (NH4)2SO4 precipitate or in 15% glycerol. Since glycolate inhibits the oxidation of glyoxylate to oxalate by glycolic acid oxidase, it is suggested that glycolic acid oxidase contributes to the synthesis of oxalate in vivo when the glyoxylate concentration is high and the glycolate concentration is low.     

Active site and loop 4 movements within human glycolate oxidase: implications for substrate specificity and drug design

Human glycolate oxidase (GO) catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and glyoxylate to oxalate, a key metabolite in kidney stone formation. We report herein the structures of recombinant GO complexed with sulfate, glyoxylate, and an inhibitor, 4-carboxy-5-dodecylsulfanyl-1,2,3-triazole (CDST), determined by X-ray crystallography. In contrast to most alpha-hydroxy acid oxidases including spinach glycolate oxidase, a loop region, known as loop 4, is completely visible when the GO active site contains a small ligand. The lack of electron density for this loop in the GO-CDST complex, which mimics a large substrate, suggests that a disordered to ordered transition may occur with the binding of substrates. The conformational flexibility of Trp110 appears to be responsible for enabling GO to react with alpha-hydroxy acids of various chain lengths. Moreover, the movement of Trp110 disrupts a hydrogen-bonding network between Trp110, Leu191, Tyr134, and Tyr208. This loss of interactions is the first indication that active site movements are directly linked to changes in the conformation of loop 4. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-hydroxy octanoate indicate that the oxidation of glycolate to glyoxylate is the primary reaction catalyzed by GO, while the oxidation of glyoxylate to oxalate is most likely not relevant under normal conditions. However, drugs that exploit the unique structural features of GO may ultimately prove to be useful for decreasing glycolate and glyoxylate levels in primary hyperoxaluria type 1 patients who have the inability to convert peroxisomal glyoxylate to glycine.     

Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.     

On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b2 (yeast L-lactate dehydrogenase).

The family of FMN-dependent, alpha-hydroxy acid-oxidizing enzymes catalyzes substrate dehydrogenation by a mechanism the first step of which is abstraction of the substrate alpha-proton (so-called carbanion mechanism). For flavocytochrome b2 and lactate oxidase, it was shown that once on the enzyme this proton is lost only slowly to the solvent (Lederer F, 1984, In: Bray RC, Engel PC, Mayhew SG, eds, Flavins & flavoproteins, Berlin: Walter de Gruyter & Co., pp 513-526; Urban P, Lederer F, 1985, J Biol Chem 260:11115-11122). This suggested the occurrence of a pKa increase of the catalytic histidine upon enzyme reduction by substrate. For flavocytochrome b2, the crystal structure indicated 2 possible origins for the stabilization of the imidazolium form of His 373: either a network of hydrogen bonds involving His 373, Tyr 254, flavin N5 and O4, a heme propionate, and solvent molecules, and/or electrostatic interactions with Asp 282 and with the reduced cofactor N1 anion. In this work, we probe the effect of the hydrogen bond network at the active site by studying proton exchange with solvent for 2 mutants: Y254F and the recombinant flavodehydrogenase domain, in which this network should be disrupted. The rate of proton exchange, as determined by intermolecular hydrogen transfer experiments, appears identical in the flavodehydrogenase domain and the wild-type enzyme, whereas it is about 3-fold faster in the Y254F mutant. It thus appears that specific hydrogen bonds to the solvent do not play a major role in stabilizing the acid form of His 373 in reduced flavocytochrome b2. Removal of the Y254 phenol group induces a pKa drop of about half a pH unit for His 373 in the reduced enzyme. Even then, the rate of exchange of the imidazolium proton with solvent is still lower by several orders of magnitude than that of a normally ionizing histidine. Other factors must then also contribute to the pKa increase, such as the electrostatic interactions with D282 and the anionic reduced cofactor, as suggested by the crystal structure.     

A highly active, soluble mutant of the membrane-associated (S)-mandelate dehydrogenase from Pseudomonas putida.

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, is a membrane-associated protein, in contrast to the more well-characterized members of this protein family including glycolate oxidase (GOX) from spinach. In a previous study [Mitra, B., et al. (1993) Biochemistry 32, 12959-12967], the membrane association of MDH was correlated to a 53 amino acid segment in the interior of the primary sequence by construction of a chimeric enzyme, MDH-GOX1, in which the membrane-binding segment in MDH was deleted and replaced with the corresponding 34 amino acid segment from the soluble GOX. Though MDH-GOX1 was soluble, it was an inefficient, nonspecific enzyme that involved a different transition state for the catalyzed reaction from that of the wild-type MDH. In the present study, it is shown that the membrane-binding segment in MDH is somewhat shorter, approximately 39 residues long. Partial or total deletion of this segment disrupts membrane localization of MDH. This segment is not important for substrate oxidation activity. A new chimera, MDH-GOX2, was created by replacing this shorter membrane-binding segment from MDH with the corresponding 20 amino acid segment from GOX. The soluble MDH-GOX2 is very similar to the wild-type membrane-bound enzyme in its spectroscopic properties, substrate specificity, catalytic activity, kinetic mechanism, and lack of reactivity toward oxygen. Therefore, it should prove to be a highly useful model for structural studies of MDH.     

Solvent and primary deuterium isotope effects show that lactate CH and OH bond cleavages are concerted in Y254F flavocytochrome b2, consistent with a hydride transfer mechanism

Yeast flavocytochrome b(2) catalyzes the oxidation of lactate to pyruvate; because of the wealth of structural and mechanistic information available, this enzyme has served as the model for the family of flavoproteins catalyzing oxidation of alpha-hydroxy acids. Primary deuterium and solvent isotope effects have now been used to analyze the effects of mutating the active site residue Tyr254 to phenylalanine. Both the V(max) and the V/K(lactate) values decrease about 40-fold in the mutant enzyme. The primary deuterium isotope effects on the V(max) and the V/K(lactate) values increase to 5.0, equivalent to the intrinsic isotope effect for the wild-type enzyme. In addition, both the V(max) and the V/K(lactate) values exhibit solvent isotope effects of 1.5. Measurement of the solvent isotope effect with deuterated lactate establishes that the primary and solvent isotope effects arise from the same chemical step, consistent with concerted cleavage of the lactate OH and CH bonds. The pH dependence of the mutant enzyme is not significantly different from that of the wild-type enzyme; this is most consistent with a requirement that the side chain of Tyr254 be uncharged for catalysis. The results support a hydride transfer mechanism for the mutant protein and, by extension, wild-type flavocytochrome b(2) and the other flavoproteins catalyzing oxidation of alpha-hydroxy acids.     

A structural model for the nucleotide binding domains of the flavocytochrome b-245 beta-chain.

NADPH is a system in phagocytic cells that generates O2- and hydrogen peroxide in the endocytic vacuole, both of which are important for killing of the engulfed microbe. Dysfunction of this oxidase results in the syndrome of chronic granulomatous disease, characterized by a profound predisposition to bacterial and fungal infections. A flavocytochrome b is the site of most of the mutations causing this syndrome. The FAD and NADPH binding sites have been located on the beta subunit of this molecule, the C-terminal half of which showed weak sequence similarity to other reductases, including the ferredoxin-NADP reductase (FNR) of known structure. This enabled us to build a model of the nucleotide binding domains of the flavocytochrome using this structure as a template. The model was built initially using a novel automatic modeling method based on distance-matrix projection and then refined using energy minimization with appropriate side-chain torsional constraints. The resulting model rationalized much of the observed sequence conservation and identified a large insertion as a potential regulatory domain. It confirms the inclusion of the neutrophil flavocytochrome b-245 (Cb-245) as a member of the FNR family of reductases and strongly supports its function as the proximal electron transporting component of the NADPH oxidase.     

Identification and characterization of HAOX1, HAOX2, and HAOX3, three human peroxisomal 2-hydroxy acid oxidases

Computer-based approaches identified three distinct human 2-hydroxy acid oxidase genes, HAOX1, HAOX2, and HAOX3, that encode proteins with significant sequence similarity to plant glycolate oxidase, a prototypical 2-hydroxy acid oxidase. The products of these genes are targeted to peroxisomes and have 2-hydroxy acid oxidase activities. Each gene displays a distinct tissue-specific pattern of expression, and each enzyme exhibits distinct substrate preferences. HAOX1 is expressed primarily in liver and pancreas and is most active on the two-carbon substrate, glycolate, but is also active on 2-hydroxy fatty acids. HAOX2 is expressed predominantly in liver and kidney and displays highest activity toward 2-hydroxypalmitate. HAOX3 expression was detected only in pancreas, and this enzyme displayed a preference for the medium chain substrate 2-hydroxyoctanoate. These results indicate that all three human 2-hydroxy acid oxidases are involved in the oxidation of 2-hydroxy fatty acids and may also contribute to the general pathway of fatty acid alpha-oxidation. Primary hyperoxaluria type 1 (PH1) is caused by defects in peroxisomal alanine-glyoxylate aminotransferase, the enzyme that normally eliminates intraperoxisomal glyoxylate. The presence of HAOX1 in liver and kidney peroxisomes and the ability of HAOX1 to oxidize glyoxylate to oxalate implicate HAOX1 as a mediator of PH1 pathophysiology.