A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxido-reductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at 1.8-A resolution. In the sulfo-form the molybdenum molybdopterin cytosine dinucleotide cofactor has a dithiolene-bound fac-[Mo, = O, = S, ---(OH2)] substructure. Bound inhibitory isopropanol in the inner compartment of the substrate binding tunnel is a model for the Michaelis complex of the reaction with aldehydes (H-C = O,-R). The reaction is proposed to proceed by transfer of the molybdenum-bound water molecule as OH- after proton transfer to Glu-869 to the carbonyl carbon of the substrate in concert with hydride transfer to the sulfido group to generate [MoIV, = O, -SH, ---(O-C = O, -R)). Dissociation of the carboxylic acid product may be facilitated by transient binding of Glu-869 to the molybdenum. The metal-bound water is replenished from a chain of internal water molecules. A second alcohol binding site in the spacious outer compartment may cause the strong substrate inhibition observed. This compartment is the putative binding site of large inhibitors of xanthine oxidase.

Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy. The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steady-state RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in E. coli. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.

Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN

Two arginine residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-containing enzymes that catalyze the oxidation of α-hydroxyacids. In the lactate oxidase from Aerococcus viridans, these residues have been changed to lysine in two single mutations and in a double mutant form. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. With all mutant forms, there are only small effects on the reactivity of the reduced flavin with oxygen. On the other hand, the efficiency of reduction of the oxidized flavin by l-lactate is greatly reduced, particularly with the R268K mutant forms. The results demonstrate the importance of the two arginine residues in the binding of substrate and its interaction with the flavin, and are consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by lysine also results in a slow conversion of the 8-CH3- substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different physical and chemical properties from those of the FMN-enzyme.

The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway

We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA12. A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains. Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified. Arabidopsis thaliana has two CYP88A genes, both of which are expressed. Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA12. Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase.

Defense Responses to Tetrapyrrole-Induced Oxidative Stress in Transgenic Plants with Reduced Uroporphyrinogen Decarboxylase or Coproporphyrinogen Oxidase Activity1

We analyzed the antioxidative defense responses of transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for uroporphyrinogen decarboxylase or coproporphyrinogen oxidase. These plants are characterized by necrotic leaf lesions resulting from the accumulation of potentially photosensitizing tetrapyrroles. Compared with control plants, the transformants had increased levels of antioxidant mRNAs, particularly those encoding superoxide dismutase (SOD), catalase, and glutathione peroxidase. These elevated transcript levels correlated with increased activities of cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD. Total catalase activity decreased in the older leaves of the transformants to levels lower than in the wild-type plants, reflecting an enhanced turnover of this photosensitive enzyme. Most of the enzymes of the Halliwell-Asada pathway displayed increased activities in transgenic plants. Despite the elevated enzyme activities, the limited capacity of the antioxidative system was apparent from decreased levels of ascorbate and glutathione, as well as from necrotic leaf lesions and growth retardation. Our data demonstrate the induction of the enzymatic detoxifying defense system in several compartments, suggesting a photosensitization of the entire cell. It is proposed that the tetrapyrroles that initially accumulate in the plastids leak out into other cellular compartments, thereby necessitating the local detoxification of reactive oxygen species.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center.

Cytochrome oxidase is a membrane protein complex that catalyzes reduction of molecular oxygen to water and utilizes the free energy of this reaction to generate a transmembrane proton gradient during respiration. The electron entry site in subunit II is a mixed-valence dinuclear copper center in enzymes that oxidize cytochrome c. This center has been lost during the evolution of the quinoloxidizing branch of cytochrome oxidases but can be restored by engineering. Herein we describe the crystal structures of the periplasmic fragment from the wild-type subunit II (CyoA) of Escherichia coli quinol oxidase at 2.5-A resolution and of the mutant with the engineered dinuclear copper center (purple CyoA) at 2.3-A resolution. CyoA is folded as an 11-stranded mostly antiparallel beta-sandwich followed by three alpha-helices. The dinuclear copper center is located at the loops between strands beta 5-beta 6 and beta 9-beta 10. The two coppers are at a 2.5-A distance and symmetrically coordinated to the main ligands that are two bridging cysteines and two terminal histidines. The residues that are distinct in cytochrome c and quinol oxidases are around the dinuclear copper center. Structural comparison suggests a common ancestry for subunit II of cytochrome oxidase and blue copper-binding proteins.

Xanthine oxidase activity associated with arterial blood pressure in spontaneously hypertensive rats

Recent evidence in vivo indicates that spontaneously hypertensive rats (SHR) exhibit an increase in oxyradical production in and around microvascular endothelium. This study is aimed to examine whether xanthine oxidase plays a role in overproduction of oxidants and thereby may contribute to hypertensive states as a consequence of the increasing microvascular tone. The xanthine oxidase activity in SHR was inhibited by dietary supplement of tungsten (0.7 g/kg) that depletes molybdenum as a cofactor for the enzyme activity as well as by administration of (−)BOF4272 [(−)-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo(1,5-α)-1,3,5-triazine-4-monohydrate], a synthetic inhibitor of the enzyme. The characteristic elevation of mean arterial pressure in SHR was normalized by the tungsten diet, whereas Wistar Koto (WKY) rats displayed no significant alteration in the pressure. Multifunctional intravital videomicroscopy in mesentery microvessels with hydroethidine, an oxidant-sensitive fluoroprobe, showed that SHR endothelium exhibited overproduction of oxyradicals that coincided with the elevated arteriolar tone as compared with WKY rats. The tungsten diet significantly repressed these changes toward the levels observed in WKY rats. The activity of oxyradical-producing form of xanthine oxidase in the mesenteric tissue of SHR was ≈3-fold greater than that of WKY rats, and pretreatment with the tungsten diet eliminated detectable levels of the enzyme activity. The inhibitory effects of the tungsten diet on the increasing blood pressure and arteriolar tone in SHR were also reproducible by administration of (−)BOF4272. These results suggest that xanthine oxidase accounts for a putative source of oxyradical generation that is associated with an increasing arteriolar tone in this form of hypertension.

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-Å resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Ω loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal “clip” domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.

Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a3 (Fe4+ = O2−) to the oxidized form (Fe3+OH−). This redox step requires the delivery of a “chemical” H+ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660–669]. The D channel is likely to be involved in the uptake of both “chemical” and “pumped” protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a3 in the K362M and E286Q mutants under aerobic steady-state turnover conditions.

Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway

Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism that perform a variety of essential functions in higher plants. Studies over the past 30 years have supported a model in which flavonoid metabolism is catalyzed by an enzyme complex localized to the endoplasmic reticulum [Hrazdina, G. & Wagner, G. J. (1985) Arch. Biochem. Biophys. 237, 88–100]. To test this model further we assayed for direct interactions between several key flavonoid biosynthetic enzymes in developing Arabidopsis seedlings. Two-hybrid assays indicated that chalcone synthase, chalcone isomerase (CHI), and dihydroflavonol 4-reductase interact in an orientation-dependent manner. Affinity chromatography and immunoprecipitation assays further demonstrated interactions between chalcone synthase, CHI, and flavonol 3-hydroxylase in lysates from Arabidopsis seedlings. These results support the hypothesis that the flavonoid enzymes assemble as a macromolecular complex with contacts between multiple proteins. Evidence was also found for posttranslational modification of CHI. The importance of understanding the subcellular organization of elaborate enzyme systems is discussed in the context of metabolic engineering.

Phylogeny of mRNA capping enzymes

The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by the sequential action of three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase. A multifunctional polypeptide containing all three active sites is encoded by vaccinia virus. In contrast, fungi and Chlorella virus encode monofunctional guanylyltransferase polypeptides that lack triphosphatase and methyltransferase activities. Transguanylylation is a two-stage reaction involving a covalent enzyme-GMP intermediate. The active site is composed of six protein motifs that are conserved in order and spacing among yeast and DNA virus capping enzymes. We performed a structure–function analysis of the six motifs by targeted mutagenesis of Ceg1, the Saccharomyces cerevisiae guanylyltransferase. Essential acidic, basic, and aromatic functional groups were identified. The structural basis for covalent catalysis was illuminated by comparing the mutational results with the crystal structure of the Chlorella virus capping enzyme. The results also allowed us to identify the capping enzyme of Caenorhabditis elegans. The 573-amino acid nematode protein consists of a C-terminal guanylyltransferase domain, which is homologous to Ceg1 and is strictly conserved with respect to all 16 amino acids that are essential for Ceg1 function, and an N-terminal phosphatase domain that bears no resemblance to the vaccinia triphosphatase domain but, instead, has strong similarity to the superfamily of protein phosphatases that act via a covalent phosphocysteine intermediate.