Tyrosinase sequencing

An interesting combination of enzymatic with non-enzymatic transformation in a one-pot three-step multiple sequence was reported by Waldmann and coworkers [82]. Phenols 125 in the presence of oxygen and enzyme tyrosinase are hydroxylated to catechols 126 which are then oxidized in situ to ortho quinones 127. These intermediates subsequently undergo a Diels-Alder reaction with inverse electron demand by reaction with different dienophiles (Table 4.19) to give endo bicyclic 1,2-diketones 128 and 129 in good yields. 

Ponnazhagan and Kwon (1992) reported a putative tissue-specific ds-element (TE-1) located at-236 bp of the mouse tyrosinase promoter. They partially purified a TE-1 binding protein (approximately 49 kDa in size), but tissue specificity remains to be confirmed by a more detailed analysis. For the human tyrosinase promoter, Shibata et al. (1992) identified a 200-bp pigment cell-specific enhancer, located between -2.0 and -1.8 kb. A minimum core sequence of 39 bp was shown to be sufficient to confer the specific activity, although other regions (not identified so far) within the 200-bp fragment are required for more efficient expression in melanoma cells (Shibata et al., 1992). 

This brief summary shows that our understanding of the basis of melanocyte-specific expression of the tyrosinase gene and the tyrosinase-related genes TRP-1 and TRP-2 is still limited. Novel approaches such as the introduction of the 70-kb-long mouse tyrosinase gene with extensive 5 flanking sequences as recently achieved (Schedl et al., 1993) allow an experimental attack of this important question. 

Kwon, B. S., Haq, A. K Pomerantz, S. H., and Halaban, R. (1987b). Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus. Proc. Natl. Acad. Sci. USA 84 7473-7477. 

Use the techniques outlined in the experimental procedure to explore two enzymes you will study in later experiments. Study the two enzymes malate dehydrogenase (Experiment 10) and tyrosinase (Experiment 5). View structures and look at amino acid sequences as you did for human a-lactalbumin. 

V Bernan, D Filpula, W Flerber, M Bibb, E Katz. The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product. Gene 37 101-110, 1985. 

The NH2-terminal amino acid sequence of the activated wild-type tyrosinase precipitated with 5% (w/v) trichloroacetic acid was SDKIHLTDD (single-letter amino acid codes), which is known to be the NH2-terminal sequence of thioredoxin including the 10 residues from the Ser2 to Aspll. Although the initiating methinine residue was encoded in the construct, it seems to have disappeared after cleavage of the protein. 

Lerch, K. (1982). Primary structure of tyrosinase from Neurospora crassa. II. Complete amino acid sequence and chemical structure of a tripeptide containing an unusual thioether. J. Biol. Chem., 257, 6414-6419. 

Copper-binding Regions of Hemocyanins and Tyrosinases. In the previous review by Lerch, the similar copper centers of hemocyanins and phenoloxidases were thoroughly discussed on the basis of sequence comparisons. Thus, the surroundings of Cu-A and Cu-B are different as already... 

Evolution of two phenoloxidases, an arthropod and molluscan type. A close relationship between phenoloxidase and hemocyantn was deduced based on their similar sequences, physico-chemical properties and similar functions. But sequence comparisons also revealed that there is not a common phenoloxidase type the enzymes found in animals, plants, and fungi are different with respect to their sequences, size, glycosylation, and activation. Two different types of tyrosinases can be distinguished based on their sequences, structure, and function. One type (m-phenoloxidase) is more related to molluscan hemocyanin with respect to the active site. The other type (a-phenoloxidase), which is very similar to arthropod hemocyanins, is found in arthropods together with hemocyanins (Figure 9). ... 

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