Streptomyces Tyrosinases

Actinomycetes are Gram-positive soil bacteria with mycelial growth. Members of the genus Streptomyces are involved in the formation and/or degradation of complex biopolymers like lignin, melanins, and humic substances (Kutzner 1968). In addition, they are important industrial sources of bioactive compounds such as antibiotics, antitumor agents, antiparasites, immunosuppressant agents, and enzymes (Anzai et al. 2008).

About 40% of Streptomyces species produce melanin-like exopigments on tyrosine-containing agar media (Fig. 13.3), which usually (but not always) correlate with the appearance of tyrosinase activity (Arai and Mikami 1972; Claus and Kutzner 1985).

Milanin Antibiotic
Fig. 13.3 Formation of melanin by a Streptomyces strain on a tyrosine-containing agar medium

Unlike most other tyrosinase-producing organisms, these bacteria secrete the enzyme into the environment, which facilitates isolation and biochemical characterization. Natural and recombinant tyrosinases have been purified from Streptomyces glaucescens (Lerch and Ettlinger 1972), Streptomyces michiganensis (Philipp et al. 1991), Streptomyces castaneoglobisporus (Kohashi et al. 2004), and Streptomyces antibioticus (Bernan et al. 1985). The enzyme from the latter species was the first tyrosinase for which a crystallographic structure could be elucidated (Matoba et al. 2006).

Tyrosinase genes from various Streptomyces species have been sequenced and translated into a protein sequence (Claus and Decker 2006). Interestingly, putative tyrosinase genes have been found in Streptomyces species that are phenotypically melanin negative (e.g., Streptomyces coelicolor), and several tyrosinase genes have been identified in some genomes (Streptomyces avermitilis).

Other bacterial tyrosinases have been detected and/or purified from the genera Vibrio (Pomerantz and Murthy 1974), Rhizobium (Mercado-Blanco et al. 1993; Piñero et al. 2007), Bacillus (Liu et al. 2004), Thermomicrobium (Kong et al. 2000), Marinomonas (López-Serrano et al. 2002, 2004), Pseudomonas (Wang et al. 2000), and Ralstonia (Hernandez-Romero et al. 2005). The presently documented molecular masses of bacterial tyrosinases range from 14 to 75 kDa; those of Streptomyces are about 30 kDa (Claus and Decker 2006).

13.2.4.1 Biochemical Properties

The typical double-enzymatic activity of tyrosinases has been demonstrated in melanin-positive Streptomyces species, whereas melanin-negative mutants lose the cresolase activity but sometimes retain some catecholase activity (Claus and Kutzner 1985). Tyrosine methylester and caffeic acid have been shown to be the best substrates for measuring both of the enzymatic activities of Streptomyces tyrosinase.

Electrophoretic characterizations have suggested that the intra- and extracellular tyrosinases from each Streptomyces species are identical, but that enzymes from different species are not (Claus and Kutzner 1985). Isoelectric focusing revealed the presence of several tyrosinase isoenzymes in some species, with their isoelectric points lying between 5.0 and 8.0. The heterogeneity of Streptomyces tyrosinases is also reflected in their different Km constants and temperature stabilities.

Apart from the essential conserved copper-binding regions, significant sequence variations among bacterial tyrosinases have been detected. Among streptomycetes, the overall relationship varies between 36 and 86% (Claus and Decker 2006).

13.2.4.2 Incorporation of Copper

The melanin operons of S. antibioticus (Katz et al. 1983; Bernan et al. 1985; Betancourt et al. 1992), S. glaucescens (Hintermann et al. 1985; Huber et al. 1985),

Streptomyces lavendulae (Kawamoto et al. 1993), and S. castaneoglobisporus (Ikeda et al. 1996) consist of two components: melC1, which encodes upstream for a small chaperon-like ("caddy") protein, and the tyrosinase structure gene melC2. Genetic and biochemical studies, predominantly with S. antibioticus, have shown that the MelC1 protein is responsible for the incorporation of copper and thus the activation of the apotyrosinase (Lee et al. 1988; Chen et al. 1992). The histidine residues of the caddy protein may serve as the copper ligands: mutational exchanges of specific histidines in the MelC1 protein resulted in significant losses of tyrosinase activity (Chen et al. 1993). The MelC1 and MelC2 proteins form stable binary complexes which can be purified by chromatographic methods. Addition of copper to the binary complexes resulted in the incorporation of two copper molecules and the release of the activated tyrosinase (Chen et al. 1992).

13.2.4.3 Induction and Secretion

Tyrosinase synthesis by S. glaucescens is surprisingly not induced by tyrosine, but by different amino acids like phenylalanine, methionine and leucine (Baumann et al. 1976). Methionine also induces the tyrosinase from S. antibioticus (Katz and Betancourt 1988; Betancourt et al. 1992). The expression of the S. castaneoglobisporus tyrosinase is favored by methionine and copper (Ikeda et al. 1996). On the other hand, the transcription of the S. michiganensis tyrosinase is induced by copper and repressed by ammonium (Held and Kutzner 1990). In chemostat experiments, oxygen was found to be a negative regulator of the tyrosinase of S. glaucescens (Wyss and Ettlinger 1981).

Although Streptomyces tyrosinases are found intra- and extracellularly, they contain no signal sequences for secretion, like all bacterial tyrosinases studied so far. The TAT pathway (twin-arginine translocation pathway) allows the transport of (metallo)proteins in their native folded conformation. Proteins secreted in this way display a characteristic twin-arginine motif between the charged N-terminus and the hydrophobic core of the leader peptide. The MelC1 "caddy" proteins have this recognition signature and are most likely transported by the TAT route, which is widely used by streptomycetes (Schaerlaekens et al. 2004). A mechanism has been proposed in which the apotyrosinase forms a binary complex with the "caddy" protein, copper is incorporated, and it is then transported across the cytoplasmic membrane (Leu et al. 1992).

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