William H. Flurkey

Sequence and structural features of plant and fungal tyrosinases.

Tyrosinases from various organisms are compared with respect to enzymatic structure, primary, secondary and tertiary structure, domain structure, Cu binding sites, maturation mechanism and activation mechanism. On the basis of these comparisons, and by using hemocyanin structure as a template, a structure model for the active site of tyrosinases is proposed.

Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase

Three cDNA clones were isolated which code for the ubiquitous chloroplast enzyme, polyphenol oxidase (PPO), from Vicia faba. Analysis of the cloned DNA reveals that PPO is synthesized with an N-terminal extension of 92 amino acid residues, presumed to be a transit peptide. The mature protein is predicted to have a molecular mass of 58 kDa which is in close agreement to the molecular mass estimated for the in vivo protein upon SDS-PAGE. Differences in the DNA sequence of two full-length and one partial cDNA clones indicate that PPO is encoded by a gene family. Analysis of the deduced amino acid sequence shows that the chloroplast PPO shares homology with the 59 kDa PPOs in glandular trichomes of solanaceous species. A high degree of sequence conservation was found with the copper-binding domains of the 59 kDa tomato PPO as well as hemocyanins and tyrosinases from a wide diversity of taxa.

Differentiation of tyrosinase and laccase using 4-hexyl-resorcinol, a tyrosinase inhibitor

Abstract Crude extracts of Agaricus bisporus and partially purified mushroom tyrosinase contained tyrosinase and laccase activities. 4-Hexylresorcinol (4HR) and salicylhydroxamic acid (SHAM) inhibited tyrosinase, but not laccase when selective substrates for tyrosinase and laccase were used in enzyme assays. Partially purified laccase from Pyricularia oryzae did not exhibit tyrosinase activity, nor was it inhibited by 4HR or SHAM. Both 4HR and SHAM selectively inhibited tyrosinase isoforms separated by native electrophoresis. At low concentrations, 4HR was a selective inhibitor of tyrosinase and could differentiate between tyrosinase and laccase activities.

Activation and alteration of plant and fungal polyphenoloxidase isoenzymes in sodium dodecylsulfate electrophoresis

Abstract Electrophoretic analysis of polyphenoloxidase isoenzymes from a variety of angiosperms and from mushroom revealed that the enzymes remain active in the presence of 0.1 % sodium dodecylsulfate. Electrophoresis in the presence of sodium dodecylsulfate allows the detection of latent enzyme forms of polyphenoloxidase, and can also convert slower migrating enzyme forms to faster migrating forms. Electrophoresis in the absence of sodium dodecylsulfate followed by incubation in the presence of sodium dodecylsulfate can also be used to detect latent forms of polyphenoloxidase. Together, these approaches provide a method for screening latent enzymes and give some insight into the mechanism of activation by sodium dodecylsulfate.

Enzyme, Protein, Carbohydrate, and Phenolic Contaminants in Commercial Tyrosinase Preparations: Potential Problems Affecting Tyrosinase Activity and Inhibition Studies

Commercial mushroom tyrosinase contains other proteins, enzymes, carbohydrates, and phenolic material besides tyrosinase. Carbohydrate and phenolic material comprise a large percentage of the powder resuspensions derived from Agaricus bisporus. Enzyme assays identified the presence of tyrosinase, laccase, beta-glucosidase, beta-galactosidase, beta-xylosidase, cellulase, chitinase, xylanase, and mannanase in the commercial tyrosinase. Protein sequencing indicated the presence of tyrosinase, a lectin, and a putative mannanase as well as 10 unidentified protein/peptides in the commercial tyrosinase preparations. Characteristics of tyrosinase isoforms were similar in two different commercial tyrosinase sources. Inhibition studies indicated that I 50 values for some tyrosinase inhibitors were different when the crude powder was compared to a partially purified tyrosinase. The presence of these contaminants has the potential to affect studies using commercial tyrosinase.

Structure and function of a virally encoded fungal toxin from Ustilago maydis: a fungal and mammalian Ca2+ channel inhibitor

BACKGROUND The P4 strain of the corn smut fungus, Ustilago maydis, secretes a fungal toxin, KP4, encoded by a fungal virus (UMV4) that persistently infects its cells. UMV4, unlike most other (non-fungal) viruses, does not spread to uninfected cells by release into the extracellular milieu during its normal life cycle and is thus dependent upon host survival for replication. In symbiosis with the host fungus, UMV4 encodes KP4 to kill other competitive strains of U. maydis, thereby promoting both host and virus survival. KP4 belongs to a family of fungal toxins and determining its structure should lead to a better understanding of the function and evolutionary origins of these toxins. Elucidation of the mechanism of toxin action could lead to new anti-fungal agents against human pathogens. RESULTS We have determined the atomic structure of KP4 to 1.9 A resolution. KP4 belongs to the alpha/beta-sandwich family, and has a unique topology comprising a five-stranded antiparallel beta-sheet with two antiparallel alpha-helices lying at approximately 45 degrees to these strands. The structure has two left-handed beta alpha beta cross-overs and a basic protuberance extending from the beta-sheet. In vivo experiments demonstrated abrogation of toxin killing by Ca2+ and, to a lesser extent, Mg2+. These results led to experiments demonstrating that the toxin specifically inhibits voltage-gated Ca2+ channels in mammalian cells. CONCLUSIONS Similarities, although somewhat limited, between KP4 and scorpion toxins led us to investigate the possibility that the toxic effects of KP4 may be mediated by inhibition of cation channels. Our results suggest that certain properties of fungal Ca2+ channels are homologous to those in mammalian cells. KP4 may, therefore, be a new tool for studying mammalian Ca2+ channels and current mammalian Ca2+ channel inhibitors may be useful lead compounds for new anti-fungal agents.

Activity, isoenzymes and purity of mushroom tyrosinase in commercial preparations

Abstract Several lots of commercial tyrosinase preparations were examined with regard to their enzyme activity, isoenzyme composition and purity. Enzyme activity toward catechol, l -dopa and tyrosine showed significant variations from lot to lot and activation by SDS. Distribution of isoenzyme forms also varied from lot to lot. Comparisons of electrophoretic and isoelectric focusing protein profiles showed considerable differences and distributions of the proteins in each sample. Tyrosinase appeared to be a minor component in each preparation when compared to a partially purified enzyme. Investigators using commercial tyrosinase should exercise caution in interpreting data due to the presence of different isoenzyme forms, their distribution in various lots, and the presence of numerous other proteins.

Isolation and characterization of a mung bean leaf polyphenol oxidase

Abstract Crude extracts from mung bean leaves, roots, stems and dark grown seedlings contained multiple forms of polyphenol oxidase (PPO) with subunit Mr values of 65 × 103, 59 × 103, 52 × 103, 47 × 103, 31 × 103 and 21 × 103. Only two of these forms were present in broken and intact leaf chloroplasts. A mature form of PPO, purified to apparent homogeneity from leaf chloroplasts, contained two proteins with subunit Mr values of 65 × 103 and 59 × 103, respectively. The purified enzyme had a pH optimum of 6.0, a pI of 5.1, and oxidized a variety of o-diphenols. The Km for l -dopa was 24 mM. o-Diphenol oxidation was inhibited by salicyl hydroxamic acid and 2,3-naphthalenediol, whereas methimazole, tropolone and 4-hexylresorcinol only resulted in partial inhibition.

Cross-reactivity of polyclonal and monoclonal antibodies to polyphenoloxidase in higher plants

Abstract Polyclonal and monoclonal antibodies against broad bean polyphenoloxidase were used to examine the cross reactivity of the enzyme in higher plants after Western blotting. Both the polyclonal and monoclonal antibodies recognized similar enzyme forms in broad beans and mung beans, but only the polyclonal antibody identified polyphenoloxidase in pea and soybean extracts. The polyclonal antibodies were able to identify inactive and/or active enzyme forms of polyphenoloxidase in the various plant sources under partially denaturing SDS-PAGE. The polyclonal antibody was also used to identify several cross-reacting proteins in five other plant species after denaturing SDS-PAGE. Under denaturing SDS-PAGE, the number and types of polyphenoloxidase identified were surprisingly similar among the eight species and were localized to proteins with M r s of 60–63 000, 43–45 000, and 34–36 000.

Purification and molecular properties of the toxin coded by Ustilagomaydis virus P4

The toxin from the P4 strain of Ustilago maydis was purified and characterized using a series of gel-filtration and ion-exchange columns. The apparent molecular weight of the purified toxin was estimated from gel electrophoresis to be 11.3 kd in the presence of 2-mercaptoethanol and 10.3 kd in the absence of 2-mercaptoethanol. Amino acid analysis indicated 12% basic amino acids, 14% acidic amino acids and 16% glycine. The toxin was also stable to filtration and repeated freezing at -20 degrees C and thawing.