The biological functions of polyamine oxidation products by amine oxidases: Perspectives of clinical applications

Summary. The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H₂O₂ and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H₂O₂ generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.     

Antimicrobial actions of dual oxidases and lactoperoxidase

The NOX/DUOX family of NADPH oxidases are transmembrane proteins generating reactive oxygen species as their primary enzymatic products. NADPH oxidase (NOX) 1–5 and Dual oxidase (DUOX) 1 and 2 are members of this family. These enzymes have several biological functions including immune defense, hormone biosynthesis, fertilization, cell proliferation and differentiation, extracellular matrix formation and vascular regulation. They are found in a variety of tissues such as the airways, salivary glands, colon, thyroid gland and lymphoid organs. The discovery of NADPH oxidases has drastically transformed our view of the biology of reactive oxygen species and oxidative stress. Roles of several isoforms including DUOX1 and DUOX2 in host innate immune defense have been implicated and are still being uncovered. DUOX enzymes highly expressed in the respiratory and salivary gland epithelium have been proposed as the major sources of hydrogen peroxide supporting mucosal oxidative antimicrobial defenses. In this review, we shortly present data on DUOX discovery, structure and function, and provide a detailed, up-to-date summary of discoveries regarding antibacterial, antiviral, antifungal, and antiparasitic functions of DUOX enzymes. We also present all the literature describing the immune functions of lactoperoxidase, an enzyme working in partnership with DUOX to produce antimicrobial substances.     

Flavoprotein oxidases: classification and applications

This review provides an overview of oxidases that utilise a flavin cofactor for catalysis. This class of oxidative flavoenzymes has shown to harbour a large number of biotechnologically interesting enzymes. Applications range from their use as biocatalysts for the synthesis of pharmaceutical compounds to the integration in biosensors. Through the recent developments in genome sequencing, the number of newly discovered oxidases is steadily growing. Recent progress in the field of flavoprotein oxidase discovery and the obtained biochemical knowledge on these enzymes are reviewed. Except for a structure-based classification of known flavoprotein oxidases, also their potential in recent biotechnological applications is discussed.     

Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78

White rot fungi efficiently degrade lignin, a complex aromatic polymer in wood that is among the most abundant natural materials on earth. These fungi use extracellular oxidative enzymes that are also able to transform related aromatic compounds found in explosive contaminants, pesticides and toxic waste. We have sequenced the 30-million base-pair genome of Phanerochaete chrysosporium strain RP78 using a whole genome shotgun approach. The P. chrysosporium genome reveals an impressive array of genes encoding secreted oxidases, peroxidases and hydrolytic enzymes that cooperate in wood decay. Analysis of the genome data will enhance our understanding of lignocellulose degradation, a pivotal process in the global carbon cycle, and provide a framework for further development of bioprocesses for biomass utilization, organopollutant degradation and fiber bleaching. This genome provides a high quality draft sequence of a basidiomycete, a major fungal phylum that includes important plant and animal pathogens.     

Restoration of peroxisomal catalase import in a model of human cellular aging

Peroxisomes play an important role in human cellular metabolism by housing enzymes involved in a number of essential biochemical pathways. Many of these enzymes are oxidases that transfer hydrogen atoms to molecular oxygen forming hydrogen peroxide. The organelle also contains catalase, which readily decomposes the hydrogen peroxide, a potentially damaging oxidant. Previous work has demonstrated that aging compromises peroxisomal protein import with catalase being particularly affected. The resultant imbalance in the relative ratio of oxidases to catalase was seen as a potential contributor to cellular oxidative stress and aging. Here we report that altering the peroxisomal targeting signal of catalase to the more effective serine-lysine-leucine (SKL) sequence results in a catalase molecule that more strongly interacts with its receptor and is more efficiently imported in both in vitro and in vivo assays. Furthermore, catalase-SKL monomers expressed in cells interact with endogenous catalase subunits resulting in altered trafficking of the latter molecules. A dramatic reduction in cellular hydrogen peroxide levels accompanies this increased peroxisomal import of catalase. Finally, we show that catalase-SKL stably expressed in cells by retroviral-mediated transduction repolarizes mitochondria and reduces the number of senescent cells in a population. These results demonstrate the utility of a catalase-SKL therapy for the restoration of a normal oxidative state in aging cells.     

Oxidation of Polyamines and Brain Injury

Several amine oxidases are involved in the metabolism of the natural polyamines putrescine, spermidine, and spermine, and play a role in the regulation of intracellular concentrations, and the elimination of these amines. Since the products of the amine oxidase-catalyzed reactions - hydrogen peroxide and aminoaldehydes - are cytotoxic, oxidative degradations of the polyamines have been considered as a cause of apoptotic cell death, among other things in brain injury. Since a generally accepted, unambiguous nomenclature for amine oxidases is missing, considerable confusion exists with regard to the polyamine oxidizing enzymes. Consequently the role of the different amine oxidases in physiological and pathological processes is frequently misunderstood. In the present overview the reactions, which are catalyzed by the different polyamine-oxidizing enzymes are summarized, and their potential role in brain damage is discussed.     

Generating disulfides with the Quiescin-sulfhydryl oxidases

The Quiescin-sulfhydryl oxidase (QSOX) family of flavoenzymes catalyzes the direct and facile insertion of disulfide bonds into unfolded reduced proteins with concomitant reduction of oxygen to hydrogen peroxide. This review discusses the chemical mechanism of these enzymes and the involvement of thioredoxin and flavin-binding domains in catalysis. The variability of CxxC motifs in the QSOX family is highlighted and attention is drawn to the steric factors that may promote efficient thiol/disulfide exchange during oxidative protein folding. The varied cellular location of these multi-domain sulfhydryl oxidases is reviewed and potential intracellular and extracellular roles are summarized. Finally, this review identifies important unresolved questions concerning this ancient family of sulfhydryl oxidases.     

Different signaling and cell death roles of heterotrimeric G protein alpha and beta subunits in the Arabidopsis oxidative stress response to ozone

Arabidopsis thaliana plants with null mutations in the genes encoding the alpha and beta subunits of the single heterotrimeric G protein are less and more sensitive, respectively, to O3 damage than wild-type Columbia-0 plants. The first peak of the bimodal oxidative burst elicited by O3 in wild-type plants is almost entirely missing in both mutants. The late peak is normal in plants lacking the Gbeta protein but missing in plants lacking the Galpha protein. Endogenous reactive oxygen species (ROS) are first detectable in chloroplasts of leaf epidermal guard cells. ROS production in adjacent cells is triggered by extracellular ROS signals produced by guard cell membrane-associated NADPH oxidases encoded by the AtrbohD and AtrbohF genes. The late, tissue damage-associated component of the oxidative burst requires only the Galpha protein and arises from multiple cellular sources. The early component of the oxidative burst, arising primarily from chloroplasts, requires signaling through the heterotrimer (or the Gbetagamma complex) and is separable from Galpha-mediated activation of membrane-bound NADPH oxidases necessary for both intercellular signaling and cell death.     

White-rot fungi and their enzymes for the treatment of industrial dye effluents

White-rot fungi produce various isoforms of extracellular oxidases including laccase, Mn peroxidase and lignin peroxidase (LiP), which are involved in the degradation of lignin in their natural lignocellulosic substrates. This ligninolytic system of white-rot fungi (WRF) is directly involved in the degradation of various xenobiotic compounds and dyes. This review summarizes the state of the art in the research and prospective use of WRF and their enzymes (lignin-modifying enzymes, LME) for the treatment of industrial effluents, particularly dye containing effluents. The textile industry, by far the most avid user of synthetic dyes, is in need of ecoefficient solutions for its colored effluents. The decolorization and detoxification potential of WRF can be harnessed thanks to emerging knowledge of the physiology of these organisms as well as of the biocatalysis and stability characteristics of their enzymes. This knowledge will need to be transformed into reliable and robust waste treatment processes.     

Dimer Interface Migration in a Viral Sulfhydryl Oxidase

Large double-stranded DNA viruses, including poxviruses and mimiviruses, encode enzymes to catalyze the formation of disulfide bonds in viral proteins produced in the cell cytosol, an atypical location for oxidative protein folding. These viral disulfide catalysts belong to a family of sulfhydryl oxidases that are dimers of a small five-helix fold containing a Cys-X-X-Cys motif juxtaposed to a flavin adenine dinucleotide cofactor. We report that the sulfhydryl oxidase pB119L from African swine fever virus (ASFV) uses for self-assembly surface different from that observed in homologs from mammals, plants, and fungi. Within a protein family, different packing interfaces for the same oligomerization state are extremely rare. We find that the alternate dimerization mode seen in ASFV pB119L is not characteristic of all viral sulfhydryl oxidases, as the flavin-binding domain from a mimivirus sulfhydryl oxidase assumes the same dimer structure as the known eukaryotic enzymes. ASFV pB119L demonstrates the potential of large double-stranded DNA viruses, which have faster mutation rates than their hosts and the tendency to incorporate host genes, to pioneer new protein folds and self-assembly modes.     

In vivo gene expression profiling of the entomopathogenic fungus Beauveria bassiana elucidates its infection stratagems in Anopheles mosquito

The use of entomopathogenic fungi to control mosquitoes is a promising tool for reducing vector-borne disease transmission.To better understand infection stratagems of insect pathogenic fungi,we analyzed the global gene expression profiling of Beauveria bassiana at 36,60,84 and 108 h after topical infection of Anopheles stephensi adult mosquitoes using RNA sequencing(RNA-Seq).A total of 5,354 differentially expressed genes(DEGs) are identified over the course of fungal infection.When the fungus grows on the mosquito cuticle,up-regulated DEGs include adhesion-related genes involved in cuticle attachment,Pthll-like GPCRs hypothesized to be involved in host recognition,and extracellular enzymes involved in the degradation and penetration of the mosquito cuticle.Once in the mosquito hemocoel,the fungus evades mosquito immune system probably through up-regulating expression of |3-l,3-glucan degrading enzymes and chitin synthesis enzymes for remodeling of cell walls.Moreover,six previous unknown SSCP(small secreted cysteine-rich proteins) are significantly up-regulated,which may serve as "effectors" to suppress host defense responses.B.bassiana also induces large amounts of antioxidant genes to mitigate host-generated exogenous oxidative stress.At late stage of infection,B.bassiana activates a broad spectrum of genes including nutrient degrading enzymes,some transporters and metabolism pathway components,to exploit mosquito tissues and hemolymph as a nutrient source for hyphal growth.These findings establish an important framework of knowledge for further comprehensive elucidation of fungal pathogenesis and molecular mechanism of Beauveria-mosqaito interactions.     

Horizontal Gene Transfer Involved in the Convergent Evolution of the Plasmid-Encoded Enantioselective 6-Hydroxynicotine Oxidases

The D- and L-specific nicotine oxidases are flavoproteins involved in the oxidative degradation of nicotine by the Gram-positive soil bacterium Arthrobacter nicotinovorans. Their structural genes are located on a 160-kbp plasmid together with those of other nicotine-degrading enzymes. They are structurally unrelated at the DNA as well as at the protein level. Each of these oxidases possesses a high degree of substrate specificity; their catalytic stereoselectivity is absolute, although they are able to bind both enantiomeric substrates with a similar affinity. It appears that the existence of these enzymes is the result of convergent evolution. The amino acid sequence of 6-hydroxy-l-nicotine oxidase (EC 1.5.3.6) as derived from the respective structural gene shows considerable structural similarity with eukaryotic monoamine oxidases (EC 1.4.3.4) but not with monoamine oxidases from prokaryotic bacteria including those of the genus Arthrobacter. These similarities are not confined to the nucleotide-binding sites. A 100-amino acid stretch at the N-terminal regions of 6-hydroxy-l-nicotine oxidase and human monoamine oxidases A possess a 35% homology. Overall, 27.0, 26.9, and 25.8% of the amino acid positions of the monoamine oxidases of Aspergillus niger (N), humans (A), and rainbow trout (Salmo gairdneri) are identical to those of 6-hydroxy-l-nicotine oxidase (Smith–Waterman algorithm). In addition, the G+C content of the latter enzyme is in the range of that of eukaryotic monoamine oxidases and definitely lower than that of the A. nicotinovorans DNA and even that of the pAO1 DNA. The primary structure of 6-hydroxy-d-nicotine oxidase (EC 1.5.3.5) does not reveal its evolutionary history as easily. Significant similarities are found with a mitomycin radical oxidase from Streptomyces lavendulae (23.3%) and a ``hypothetical protein' from Mycobacterium tuberculosis (26.0%). It is proposed that the plasmid-encoded gene of 6-hydroxy-l-nicotine oxidase evolved after horizontal transfer from an eukaryotic source. Received: 6 March 1998 / Accepted: 15 July 1998     

NADPH oxidases are involved in differentiation and pathogenicity in Botrytis cinerea

Nicotinamide adenine dinucleotide (NADPH) oxidases have been shown to be involved in various differentiation processes in fungi. We investigated the role of two NADPH oxidases in the necrotrophic phytopathogenic fungus, Botrytis cinerea. The genes bcnoxA and bcnoxB were cloned and characterized; their deduced amino acid sequences show high homology to fungal NADPH oxidases. Analyses of single and double knock-out mutants of both NADPH oxidase genes showed that both bcnoxA and bcnoxB are involved in formation of sclerotia. Both genes have a great impact on pathogenicity: whereas bcnoxB mutants showed a retarded formation of primary lesions, probably due to an impaired formation of penetration structures, bcnoxA mutants were able to penetrate host tissue in the same way as the wild type but were much slower in colonizing the host tissue. Double mutants showed an additive effect: they were aberrant in penetration and colonization of plant tissue and, therefore, almost nonpathogenic. To study the structure of the fungal Nox complex in more detail, bcnoxR (encoding a homolog of the mammalian p67(phox), a regulatory subunit of the Nox complex) was functionally characterized. The phenotype of DeltabcnoxR mutants is identical to that of DeltabcnoxAB double mutants, providing evidence that BcnoxR is involved in activation of both Bcnox enzymes.     

Ancestral gene fusion in cellobiose dehydrogenases reflects a specific evolution of GMC oxidoreductases in fungi

Cellobiose dehydrogenases (CDHs) are extracellular hemoflavoenzymes that are thought to be involved in the degradation of two of the most abundant biopolymers in the biosphere, cellulose and lignin. To date, these enzymes, consisting of a cytochrome domain and a flavin domain, have been detected and sequenced exclusively in the kingdom of fungi. Independent phylogenetic analyses of two distinct domains of CDH genes reveal that they evolved in parallel as fused genes. Whereas the cytochrome domains are unique sequence motifs, the flavin domains clearly belong to the glucose-methanol-choline (GMC) oxidoreductase family--an evolution line of widespread flavoproteins extending from the Archae to higher eukaryotes. The most probable unrooted phylogenetic tree obtained from our analysis of 52 selected GMC members reveals five principal evolutionary branches: cellobiose dehydrogenase, cholesterol oxidase (COX), hydroxynitrile lyase, alcohol oxidase (AOX)/glucose oxidase (GOX)/choline dehydrogenase, and a branch of dehydrogenases with various specificities containing also an Archaeon open reading frame (ORF). Cellobiose dehydrogenases cluster with cholesterol oxidases and the clade of various specificities, whereas hydroxynitrile lyases are closely related to glucose oxidases, alcohol oxidases, and choline dehydrogenases. The results indicate that the evolutionary line from a primordial GMC flavoprotein to extant cellobiose dehydrogenases was augmented after an early acquisition of the cytochrome domain to form two distinct branches for basidiomycetes and ascomycetes. One ascomycetous evolutionary line of CDHs has acquired a carbohydrate-binding module (CBM) of type 1, the sequence of which is similar to that of corresponding domains in several glycosidases. This is the first attempt towards a comprehensive phylogenetic analysis of cellobiose dehydrogenases.     

Functional importance of lysyl oxidase family propeptide regions

The lysyl oxidase family of proteins is primarily known for its critical role in catalyzing extracellular oxidative deamination of hydroxylysine and lysine residues in collagens, and lysine residues in elastin required for connective tissue structure and function. Lysyl oxidases have additional important biological functions in health and disease. While the enzyme domains are highly conserved, the propeptide regions are less uniform, and have biological activity, some of which are independent of their respective enzymes. This review summarizes what has been published regarding the functions of the propeptide regions of this family of proteins in the context of extracellular matrix biosynthesis, fibrosis and cancer biology. Although much has been learned, there is a need for greater attention to structure/function relationships and mechanisms to more fully understand these multifunctional proteins.     

Autocatalysed oxidative modifications to 2-oxoglutarate dependent oxygenases

Ferrous iron and 2-oxoglutarate-dependent oxygenases and related enzymes catalyse a range of oxidative reactions, possibly the widest of any enzyme family. Their catalytic flexibility is proposed to be related to their nonhaem iron-binding site, which utilizes two or three protein-based ligands. A possible penalty for this flexibility is that they may be more prone to oxidative damage than the P450 oxidases, where the iron is arguably located in a more controlled environment. We review the evidence for autocatalysed oxidative modifications to 2-oxoglutarate-dependent oxygenases, including the recently reported studies on human enzymes, as well as the oxidative fragmentations observed in the case of the plant ethylene-forming enzyme (1-aminocyclopropane-1-carboxylic acid oxidase).     

Purine metabolism in B-cell lymphocytic leukemia: a microarray approach

B-cell chronic lymphocytic leukemia (B-CLL) is an adult-onset highly heterogeneous malignancy characterized by a cells resistance to apoptosis rather than to highly proliferative cells. In previous research, we evidenced an imbalance of purine metabolism in B-CLL cells. Since the extracellular adenosine has been proved to induce apoptosis via A2b receptor, enzymes involved in adenosine metabolism could play an important role in apoptosis resistance of B-CLL cells. We prepared a microarray chip for the analysis of 50 selected genes that could be of interest in B-CLL: enzymes of purine de-novo, salvage and catabolic pathway, oxidative stress enzymes, and apoptotis-related proteins. Preliminary results identify many genes of purine metabolism that exhibit low or high expression, while genes involved in signal transduction and apoptosis exhibit lower alterations even if of remarkable interest. This application of microarray technique seems promising and at least a subset of these genes will be valid candidates for further studies.     

Purine Metabolism in B-Cell Lymphocytic Leukemia: A Microarray Approach

B-cell chronic lymphocytic leukemia (B-CLL) is an adult-onset highly heterogeneous malignancy characterized by a cells resistance to apoptosis rather than to highly proliferative cells. In previous research, we evidenced an imbalance of purine metabolism in B-CLL cells. Since the extracellular adenosine has been proved to induce apoptosis via A2b receptor, enzymes involved in adenosine metabolism could play an important role in apoptosis resistance of B-CLL cells. We prepared a microarray chip for the analysis of 50 selected genes that could be of interest in B-CLL: enzymes of purine de-novo, salvage and catabolic pathway, oxidative stress enzymes, and apoptotis-related proteins. Preliminary results identify many genes of purine metabolism that exhibit low or high expression, while genes involved in signal transduction and apoptosis exhibit lower alterations even if of remarkable interest. This application of microarray technique seems promising and at least a subset of these genes will be valid candidates for further studies.