Plant copper oxidases

A fourth possibility is the generation of H202 via oxidation of putrescine (butane-1,4-diamine 2.56). This reaction is catalyzed by copper amine oxidase (E.C. 1.4.3.6). Copper amine oxidases are homodimers in which each unit contains a copper ion and a 1,3,5-trihydroxyphenylalanine quinine co-factor. In plants copper amine oxidases generally oxidize putrescine to 4-aminobutanal (2.57). This latter compound undergoes spontaneous cyclization to A1 pyrroline (2.58), ammonia, and H202, as shown in Figure 2-12 (Medda et al., 1995). 

Medda, R., Padiglia, A., and Floris, G., 1995, Plant copper amine oxidases. Phytochemistry 39 ln9. 

This paper summarizes briefly the physicochemistry and enzymology of plant copper oxidases with particular emphasis on polyphenol oxidase and laccase. A brief comparative discussion of other naturally occurring copper proteins and artificial copper proteins is appropriate when discussing the physicochemistry of the copper site itself. In the case of the copper proteins listed in Table I, we know a great deal more about the copper site than about the physicochemistry of the rest of the protein molecule. This is primarily a result of the availability of sophisticated spectroscopic techniques such as optical spectroscopy (both absorption and circular dichroism) and electron spin resonance which are applicable to the electronic transitions of the copper ion. On the other hand, protein chemistry has progressed more slowly. Many of the proteins are large and complex multisubunit enzymes, difficult to purify, and often unstable. There are several excellent reviews on this group of proteins (59, 60, 61, 62). 

Of the group of plant copper amine oxidases, the enzymes from pea and lentil have been studied thoroughly as regards their substrate and inhibitory properties [116]. An acetylenic analogue of the substrate putrescine, 2-butyne-1,4-diamine (DABI), has been described as a typical... 

Multicopper Oxidases (Blue Copper Oxidases) Ascorbate Oxidase, Ceruloplasmin, and Laccase. The multicopper oxidases (MCOs) are important enzymes, which are found in many plants (lignin formation), fungi (lignin degradation and detoxification), bacteria, as well as humans (ferroxidase activity) (13). MCOs catalyze the four-electron reduction of O2 to two waters with the electrons coming firom one-electron oxidation of four substrate molecules. The latter are organic reductants for ascorbate oxidase (AO) (32) and laccase (Lc) (130), and a metal ion (ferrous ion) for ceruloplasmin (Cp) (33) (Scheme 9). 

Ascorbic acid is distributed primarily in the plant kingdom, where ascorbic oxidase, a copper-containing, four-electron-transferring oxidase (cf. Chapt. X-7), is also found. Rats and many other mammals are able to synthesize ascorbic acid they do not depend on a supplementation of the vitamin. ... 

Copper is one of the twenty-seven elements known to be essential to humans (69—72) (see Mineral nutrients). The daily recommended requirement for humans is 2.5—5.0 mg (73). Copper is probably second only to iron as an oxidation catalyst and oxygen carrier in humans (74). It is present in many proteins, such as hemocyanin [9013-32-3] galactose oxidase [9028-79-9] ceruloplasmin [9031 -37-2] dopamine -hydroxylase, monoamine oxidase [9001-66-5] superoxide dismutase [9054-89-17, and phenolase (75,76). Copper aids in photosynthesis and other oxidative processes in plants. 

Polyphenol oxidase occurs within certain mammalian tissues as well as both lower (46,47) and higher (48-55) plants. In mammalian systems, the enzyme as tyrosinase (56) plays a significant role in melanin synthesis. The PPO complex of higher plants consists of a cresolase, a cate-cholase and a laccase. These copper metalloproteins catalyze the one and two electron oxidations of phenols to quinones at the expense of 02. Polyphenol oxidase also occurs in certain fungi where it is involved in the metabolism of certain tree-synthesized phenolic compounds that have been implicated in disease resistance, wound healing, and anti-nutrative modification of plant proteins to discourage herbivory (53,55). This protocol presents the Triton X-114-mediated solubilization of Vida faba chloroplast polyphenol oxidase as performed by Hutcheson and Buchanan (57). 

Copper oxidases are widely distributed in nature, and enzymes from plants, microbes, and mammals have been characterized (104,105). The blue copper oxidases, which include laccases, ascorbate oxidases, and ceruloplasmin, are of particular interest in alkaloid transformations. The principle differences in specificity of these copper oxidases are due to the protein structures as well as to the distribution and environment of copper(II) ions within the enzymes (106). While an in vivo role in metabolism of alkaloids has not been established for these enzymes, copper oxidases have been used in vitro for various alkaloid transformations. 

Rea G, Metoui O, Infantino A, Federico R, Angelini R (2002) Copper amine oxidase expression in defense responses to wounding and Ascochyta rabiei invasion. Plant Physiol 128 865-875 Roeder V, Collen J, Rousvoal S, Corre E, Leblanc C, Boyen C (2005) Identification of stress gene transcripts in Laminaria digitata (Phaeophyceae) protoplast cultures by Expressed Sequence Tag analysis. J Phycol 41 1227-1235... 

Laccase is perhaps the metallo-enzyme most widely used for this aim. Laccases are a family of multicopper ( blue copper ) oxidases widely distributed in nature Many laccases have fungal origin, while others are produced in plants. They contain four Cu(II) ions, and catalyse the one-electron oxidation of four molecules of a reducing substrate with the concomitant four-electron reduction of oxygen to water . In view of their low redox potential, which is in the range of 0.5-0.8 V vs. NHE depending on the fungal source laccases typically oxidize phenols (phenoloxidase activity) or anilines. 

The multi-copper oxidases include laccase, ceruloplasmin, and ascorbate oxidase. Laccase can be found in tree sap and in fungi ascorbate oxidase, in cucumber and related plants and ceruloplasmin, in vertebrate blood serum. Laccases catalyze oxidation of phenolic compounds to radicals with a concomitant 4e reduction of O2 to water, and it is thought that this process may be important in the breakdown of lignin. Ceruloplasmin, whose real biological function is either quite varied or unknown, also catalyzes oxidation of a variety of substrates, again via a 4e reduction of O2 to water. Ferroxidase activity has been demonstrated for it, as has SOD activity. Ascorbate oxidase catalyzes the oxidation of ascorbate, again via a 4e reduction of O2 to water. Excellent reviews of these three systems can be found in Volume 111 of Copper Proteins and Copper Enzymes (Lontie, 1984). 

Polyphenol oxidase (PPO) (EC 1.14.18.1 monophenol monooxygenase [tyrosinase] or EC 1.10.3.2 0-diphenol 02-oxidoreductase) is one of the more important enzymes involved in the formation of black tea polyphenols. The enzyme is a metallo-protein thought to contain a binudear copper active site. The substance PPO is an oligomeric particulate protein thought to be bound to the plant membranes. The bound form of the enzyme is latent and activation is likely to be dependent upon solubilization of the protein (35). PPO is distributed throughout the plant (35) and is localized within in the mitochondria (36), the cholorplasts (37), and the peroxisomes (38). Using antibody techniques, polyphenol oxidase activity has also been localized in the epidermis palisade cells (39). Reviews on the subject of PPO are available (40—42). 

Catalases and peroxidases. Many iron and copper proteins do not bind 02 reversibly but "activate" it for further reaction. We will look at such metallo-protein oxidases in Chapter 18. Here we will consider heme enzymes that react not with 02 but with peroxides. The peroxidases,1943 which occur in plants, animals, and fungi, catalyze the following reactions (Eq. 16-6,16-7) ... 

Copper was recognized as nutritionally essential by 1924 and has since been found to function in many cellular proteins.470-474 Copper is so broadly distributed in foods that a deficiency has only rarely been observed in humans.4743 However, animals may sometimes receive inadequate amounts because absorption of Cu2+ is antagonized by Zn2+ and because copper may be tied up by molybdate as an inert complex. There are copper-deficient desert areas of Australia where neither plants nor animals survive. Copper-deficient animals have bone defects, hair color is lacking, and hemoglobin synthesis is impaired. Cytochrome oxidase activity is low. The protein elastin of arterial walls is poorly crosslinked and the arteries are weak. Genetic defects in copper metabolism can have similar effects. 

Two ascorbate radicals can react with each other in a disproportionation reaction to give ascorbate plus dehydroascorbate. However, most cells can reduce the radicals more directly. In many plants this is accomplished by NADH + H+ using a flavoprotein monodehydroascorbate reductase.0 Animal cells may also utilize NADH or may reduce dehydroascorbate with reduced glutathione.CC/ff Plant cells also contain a very active blue copper ascorbate oxidase (Chapter 16, Section D,5), which catalyzes the opposite reaction, formation of dehydroascorbate. A heme ascorbate oxidase has been purified from a fungus. 11 1 Action of these enzymes initiates an oxidative degradation of ascorbate, perhaps through the pathway of Fig. 20-2. 

COPPER (In Biological Systems), The activity of copper in plant metabolism manifests itself in two forms 11) synthesis of chlorophyll, and 12) activity of enzymes. In leaves, most of the copper occurs in close association with chlorophyll, but little is known of ns rale in chlorophyll synthesis, other than the presence of cupper is required. Copper is a definite constituent of several enzymes catalyzing oxidation-reduction reactions (oxidases), in which the activity is believed to be due to the shuttling of copper between the +1 and +2 oxidalicm states,... 

Cytochrome oxidase (cytochrome aa3) represents the most important cytochrome of the a class. This is the terminal oxidase used in animals, plants, yeasts, algae and some bacteria. It contains two copper centres, giving four redox groups in total. This oxidase is discussed with other cytochromes that have a terminal oxidase function in Sections 62.1.12.4 and 62.1.12.5. These are cytochromes o, d and cd,. The oxidases fed719 and ax are not included in that discussion. The situation regarding cytochrome ax has been confused, partly due to uncertainty in the definition of this cytochrome. In some respects, the properties of cytochrome ax resemble those of mitochondrial and bacterial aa3. It functions as a terminal oxidase in some bacteria,720 but its role in E. coli is unknown. A soluble fraction from disrupted E. coli cells grown anaerobically on glycerol and fumarate contains a hemoprotein similar to cytochrome ax, which has catalase and peroxidase activity.721... 

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