MINIREVIEWBacterial tyrosinases
Introduction
Tyrosinases (E.C. 1.14.18.1) are copper-containing enzymes which are ubiquitously distributed in nature [50], [60], [84]. They are essential for the formation of melanin [7], [65], [70] and various other functions. In plants, sponges and many invertebrates, they are important components of wound healing and the primary immune response [9], [62], [84]. In arthropods they are also involved in sclerotization of the cuticle after molting or injury. In mammals tyrosinases are found in melanocytes of the retina and skin [28].
Tyrosinases use molecular oxygen to catalyse two different enzymatic reactions [21], [48], [50], [77], [84]: (i) the ortho-hydroxylation of monophenols to o-diphenols (monophenolase, cresolase acticity) and (ii) the oxidation of o-diphenols to o-quinones (diphenolase, catecholase activity). The reactive quinones polymerize non-enzymatically to the macromolecular melanins (Fig. 1). It should be noted that often the name phenoloxidase is used in literature, which summarizes tyrosinases, catecholoxidases and laccases as well. Because of their overlapping substrate specificities, a positive test for catecholase activity does not necessarily mean that the enzymes exhibit cresolase activity.
In spite of intensive biochemical investigations since decades, there exist only limited informations about the protein structure and the exact reaction mechanisms. Some reasons for these deficits are difficulties in purification of sufficiently high amounts of tyrosinases from eukaryotic sources due to low enzyme concentrations, contamination with pigments, occurrence of isoenzymes or post-translational modifications.
The copper binding sites of tyrosinases share a high sequence homology with the haemocyanins, the oxygen carrier proteins of the molluscs and arthropods [20], [22], [23], [40], [84], [85]. During evolution, a functional change of this protein family has been proposed: from enzymatic oxygen detoxification towards oxygen transport [40].
The common feature is a ‘type 3 copper centre’, a diamagnetic spin-coupled copper pair [21], [48], [50], [77], [84]. Each of the two metal atoms, CuA and CuB, of the active site are coordinated by three conserved histidines which are located in a ‘four α-helix bundle’. During the catalytic cycle the ‘type 3 copper centre’ can adopt different functional forms: the oxy-state [Cu(II)–O22−–Cu(II)], deoxy-state [Cu(I) Cu(I) ], half-met state [Cu(I) Cu(II)] and the met state [Cu(II)–OH−–Cu(II)]. In the latter case the two copper atoms are bridged by hydroxo ions. The valences of the two copper atoms change from Cu(I) to Cu(II), which can be followed spectroscopically. In the oxy-state the molecular oxygen is reversibly bound as a peroxide between the two copper atoms in a ‘side-on’ conformation (Fig. 5). In the absence of any substrate more than 85% of the enzyme is in the met state, which can be regarded as the resting form of tyrosinase. According to current conceptions, both, the met- and the oxy-state of tyrosinases enable the diphenoloxidase activity, whereas the monohydroxylase reaction requires the oxy-state.
Although much work on bacterial tyrosinases was published since decades, most studies did not concentrate on structural topics. Nevertheless, tyrosinases from microorganisms may be helpful to get more insight in the common architecture of these enzymes and especially in the complicated monohydroxylase reaction [5], [6], [79], [80], [83]. This review will focus on Streptomyces tyrosinases in the light of recent results on ‘type 3 copper’ proteins in general.
Section snippets
Streptomyces tyrosinases and melanization
Actinomycetes are Gram-positive soil bacteria with mycelious growth. Members of the genus Streptomyces are involved in the formation and/or degradation of complex biopolymers like lignin, melanins, and humic substances [47]. In addition, they are important industrial sources of antibiotics and other secondary metabolites.
About 40% of Streptomyces species produce melanin-like exopigments on tyrosine-containing agar media, which mostly but not always correlate with the appearance of an
Purification
The first bacterial tyrosinases have been purified from cell extracts of Streptomyces nigrifaciens [63] and Streptomyces glaucescens [51]. Unlike most eukaryotic tyrosinases, the active form of the S. glaucescens protein is a monomer, without tendency of concentration-dependent aggregation as shown by analytical ultracentrifugation. The enzyme has a molecular mass of 29,100 Da in SDS-PAGE and its maximum activity at pH 6.8. The extracellular tyrosinase of S. glaucescens was isolated 10 years
Regulation
For most bacterial tyrosinases it is not known whether they are produced constitutively or inducibly. Tyrosinase synthesis by S. glaucescens is surprisingly not induced by tyrosine, but by other amino acids like phenylalanine, methionine and leucine [2]. Methionine is also the inducer of the tyrosinase from S. antibioticus [4], [41]. The expression of the S. castaneoglobisporus tyrosinase is favoured by methionine and copper [38]. Otherwise, the transcription of the S. michiganensis tyrosinase
Spectroscopic characteristics
Intensive spectroscopic studies have revealed the surrounding of the two copper ions in tyrosinase. Electron-spin-resonance (ESR) and Raman spectroscopy classify tyrosinase as a coupled binuclear copper protein (type 3 copper) [50], [77], [79], [80], [83]. The paramagnetic [Cu(I) Cu(II)] half-met state of the dinuclear copper centre of the tyrosinase from S. antibioticus has been analysed by pulsed ESR and hyperfine sublevel correlation spectroscopy in the presence and absence of inhibitors
mel genes
The frequent occurrence of a melanin-negative phenotype in S. glaucescens and Streptomyces reticuli has been attributed to insertion elements [36], [76]. In the potato pest Streptomyces scabies and in Rhizobium meliloti, the tyrosinase genes are plasmid located [31], [61]. Genetic studies with melanin-negative Streptomyces mutants revealed a polycistronic organization of the chromosomal melC operon (Fig. 2). Besides the structure gene for the tyrosinase (melC2), at least one more upstream
Amino acid composition
Alanine is the dominating amino acid in the prokaryotic tyrosinase sequences. In Gram-positive bacteria there are also high amounts of charged residues (arginine, aspartate). Arginine residues favour not only the formation of salt bridges, but may simultaneously bind to aromatic stacked rings [28].
Cysteine is missing in the tyrosinases of streptomycetes except in those of Streptomyces griseus and in one of three putative tyrosinase sequences identified in the genome of Streptomyces avermitilis.
Incorporation of copper
The melanin operon of S. antibioticus [3], [4], [42], S. glaucescens [36], [37], Streptomyces lavendulae [43] and S. castaneoglobisporus [39] consists of two parts: melC1 which codes for a small helper protein and the tyrosinase structure gene melC2 (Fig. 2). Genetic and biochemical studies predominantly with S. antibioticus, have shown that the MelC1 protein is responsible for incorporation of copper and thus for activation of the apotyrosinase [10], [49]. The histidines of the activating
Secretion
In prokaryotes (bacteria and archaea) transport of extracellular proteins across the cytoplasma membrane is mainly managed by the universal secretion pathway (sec-pathway) [68]. Specific signal sequences of the proteins are recognized by the SRP (signal recognition particle) and directed to the secretion apparatus. Although Streptomyces tyrosinases are found intra- and extracellular, they contain like all bacterial tyrosinases studied so far, no signal sequences. However, as shown in Fig. 4,
Laccases
Melanization in Streptomyces and other microrganisms is not only catalysed by tyrosinases but also via different pathways and enzymes [7], [8], [65]. Laccases (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductases) are multi-copper proteins that use molecular oxygen to oxidize various aromatic and non-aromatic compounds. [13], [77]. Until recently, laccases have been only found in eukaryotes (fungi, higher plants, insects) but now there is strong evidence for their wide distribution in prokaryotes
Physiological importance and applications
In spite of their ubiquitous distribution in nature and intensive biochemical investigations, the biological functions of bacterial tyrosinases are not fully understood. In streptomycetes, tyrosine is neither best substrate nor inducer of the tyrosinases. The best-documented function of the enzyme is restricted to the formation of melanins. The dark pigments protect the bacterial cells and spores against UV radiation [7], [65], [71], [72]. For instance, the expression of the mel gene from
Relationship to other ‘type 3 copper’ proteins
Whereas a lot of information about genes, sequences, mutations and kinetics is available for Streptomyces tyrosinases, not much is known about their structure. The comparison with other ‘type 3 copper’ proteins such as haemocyanins may help to get a funded idea [20], [21], [22], [23], [33], [40], [48], [66], [79], [80], [85], Recently it was shown that haemocyanins exhibit a tyrosinase activity after specific activation [20], [22], [23], [40]. Consequently, our present knowledge of tyrosinase
Acknowledgements
H.C. wants to dedicate this article to Prof. Dr. H.J. Kutzner. The modelled structure of Streptomyces tyrosinase was kindly provided by Thorsten Schweikardt and Uwe Salzbrunn (Institute for Molecular Biophysics, University of Mainz).
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