Review

Oxidative Metabolism of Tyrosine in Insects

Martin G. Peter

Institut für Organische Chemie und Biochemie der Rheinischen

Friedrich-Wilhelms-Universität, Gerhard-Domagk-Str. 1, D-5300 BONN 1

1. Introduction

Insects utilise tyrosine, inter alia, for biosynthesis of biogenic amines, melanin pigments, and sclerotins. This review dicusses the enzymatic pathways, considering also differences in the corresponding pathways in vertebrates. Only a general outline can be given here and many details of the regulation of enzyme activities as well as differences in various insect species have to be obmitted. For in depth information, the reader is referred to the literature cited and references given therein.

2. Biosynthesis of biogenic amines

The distribution of neurotransmitters in the nervous system of insects has been studied in much detail (1,2). In contrast, little is known on the enzymes of their biosynthesis. Tyrosine hydroxylase (EC 1.14.16.1) occurs in vertebrates as a pterin and Fe2+ dependent rate limiting enzyme. It was long believed that the enzyme is absent from insects. However, more recent studies have demonstrated the occurrence of Dopa and tyrosine hydroxylase in Drosophila melanogaster (for review, see (3)). Low amounts of DOPA have been found also in the CNS of the tobacco hornworm, Manduca sexta, at a narrowly defined developmental stage (4).

Dopa is decarboxylated to dopamine by a rather specific DOPA decarboxylase as was shown first in locust brain (5). Besides, decarboxylation of tyrosine leads to tyramine which then is either ß-hydroxylated to (R)-octopamine or ring hydroxylated to dopamine. Apparently, any monophenolic phenethylamine can be converted to a diphenol. Likewise, the side chain of either a mono- or a diphenol can be ß-hydroxylated. However, due to the paucity of data on the enzymes and their specificity, it is sometimes difficult to judge whether a particular in vitro transformation is also physiologically significant. The amounts of noradrenaline in insect CNS are less than 10% of dopamine (6). The configuration of noradrenaline in insect nervous tissue has not been reported though stereochemical arguments are most critical with respect to the mechanism of ß-hydroxylation (see section 4).

The phenolic phenethylamines are N-acylated and/or O-glucosylated (4,7). Eventually, these conjugates represent storage forms of neurotransmitters or intermediates of monoamine catabolism.

3. Biosynthesis of melanins

In contrast to the situation in vertebrates, insect melanins are always extracellular pigments. Melanin is formed in the blood of insects after wounding or invasion by pathogenic microorganisms. As the polymeric pigment has blood clotting properties and encapsulates pathogens, it is involved in wound healing and in immune response.

The pathway of eumelanin biosynthesis in insect haemolymph is similar to that in in vertebrates. L-Tyrosine is hydroxylated to DOPA by a Cu2+ dependent monophenol mono-oxygenase (tyrosin-ase, EC 1.14.18.1) which is localized in the hemocytes of the hemolymph. The same enzyme has also diphenoloxidase (o-diphenol: O2 oxidoreductase, EC 1.10.3.1) activity. Thus, it should be expected that tyrosine hydroxylation by tyrosinase is followed immediately by oxidation of DOPA to dopachrome. However, insect haemolymph also contains a highly specific DOPA decarboxylase (EC 4.1.1.28) which is under control of the steroid hormone 20-hydroxyecdysone. Wounding activates both, DOPA decarboxylase and phenoloxidase, and it was concluded that the melanin involved in wound healing is derived from dopamine (8). Degradation of insect melanins yields dihydroxyindole and pyrrole carboxylic acids, but not dihydroxyindole carboxylic acid (review: (9)) and thus indicates that dopamine and dihydroxyindole, rather than DOPA and dihydroxyindole carboxylic acid are the precursors for insect melanins.

A second source of eumelanin in insects is the exocuticle, where the pigment is often found in the form of granules (9). Since cuticle is virtually devoid of cysteine, the pigment must be of the eumelanin type. Again, the precursor is dopamine and pigmentation is under endocrine control (8). The tyrosinase from pharate pupal cuticle of Manduca sexta differs in substrate specificity from vertebrate and mushroom tyrosinases. It converts tyrosine and tyramine to DOPA and dopamine and oxidizes diphenols to o-quinones with Vmax/Km values decreasing in the order N-ß-alanyldopamine > N-acetyldopamine > dopamine > dihydroxyindole > DOPA (10). In addition, the cuticle contains a proteineous DOPA quinone conversion factor which accelerates the decarboxylation of dopachrome to dihydroxyindole.

4. Biosynthesis of sclerotins

Sclerotins are defined as chemically modified proteineous components of insoluble and stiff skeletal structures. In insects, they occur in the ootheca of cockroaches and in the exo-skeleton. Here, we will consider the latter only. Briefly, the process of "sclerotization" of insect cuticle is based on oxidative reactions of N-acyldopamines with proteins of mostly unknown structure and with chitin. Other diphenols may be involved. Some recent reviews on cuticle sclerotization are available (11,12).

Hydroxylation of tyrosine by tyrosinase is followed by decarboxylation of DOPA to dopamine. Insects have evolved regulatory mechanisms that divert dopamine from the melanin to the sclerotization pathway by N-acylation with acetyl and ß-alanyl residues in hemocytes and epidermis. Storage forms of the diphenolic compounds are O-glucosides, O-phosphates and O-sulfates. Free dopamine and N-acyldopamines are translocated into the cuticle. The participating enzymes are under endocrine control.

It is generally believed that the diphenoloxidases required for oxidation of the catechols in the cuticle are the same enzymes as those found in the haemolymph. They occur also in cuticle as proenzymes and are activated at the onset of sclerotization under control of 20-hydroxyecdysone. However, the picture is more complex, since, in addition to tyrosinase (EC 1.14.18.1) and o-diphenol oxidase (EC 1.10.3.1), also laccases (EC 1.10.3.2) and peroxidases (EC 1.11.1.7) may occur in cuticle.

Insect cuticles also contain N-acetylnoradrenaline. First indications for a fundamental difference in the mechanism of side chain hydroxylation in CNS and cuticle were suggested by stereochemical arguments. From in vitro oxidation mixtures of N-acetyldopamine and insect cuticle, racemic N-acetylnoradrenaline was obtained. This is consistent with a rearrangement of 4-alkyl-o-quinones to p-quinone methides and non-stereoselective addition of water to the latter, rather than with hydroxylation by a mono-oxygenase of the dopamine ß-hydroxylase type (EC 1.14.17.1) (13,14). More recently, the o-quinone-p-quinone methide rearrangement was claimed to be accelerated by a tautomerase (EC not assigned) (15). This enzyme activity was also found in haemolymph of the fly Sarcophaga bullata (16). Furthermore, the rearrangement is observed upon oxidation of N-acyldopamines with tyrosinases and laccases from cuticles of various insects (17,18,19), and, to a low extent, with mushroom tyrosinase (13).

Eventually, the p-quinone methide generated from N-acetyldopamine-quinone, but not from N-ß-alanyldopamine-quinone, may be further rearranged by yet another enzyme to the corresponding a,ß-dehydro-N-acetyldopamine (20). Thus, the pattern of enzymatic diphenol oxidation in insect cuticle is more complex than that of melanogenesis, for which the minimum enzymatic requirement is tyrosinase, or of lignin biosynthesis in plants which requires principally peroxidase only (for review, see (21)).

Much progress has been made recently with respect to the mechanisms of oxidative polymerization of the catechols and structural protein modifications by reactive quinonoid intermediates (for review, see (21)). Linkage of imidazole-Nt of histidine to the ring position of a dopamine metabolite is formed by 1,4-Michael-type reaction to the o-quinone, and to the ß-carbon by 1,6-conjugate addition to the quinone methide, as is evident from CP-MAS NMR studies (22,23) and from in vitro reactions (24,25). Likewise, primary aliphatic amines, such as ß-alanine and the e-amino group of lysine may add to the ring and to the side chain of N-acetyldopamine (26). Polymerization of reactive intermediates via the side chain leads to benzodioxane oligomers (27) which may also be connected to protein residues (25). Also, biphenyltetrols (28,29), dibenzofuranes (29) and phenoxazines (30,31) were found recently. Crosscoupling of peptidic tyrosine residues with N-acylcatecholamines results in mixed type melanin-like materials (32).

Many variations exist in the colour of insect cuticles. Current theory holds that, as far as diphenolic precursors are involved, the colouration of cuticles is correlated with the nature of the precursors: black results from dopamine, brown from N-ß-alanyldopamine, and light or colourless from N-acetyldopamine, where simultaneous participation of all types are possible. Genetic lack of N-acylation or transport of ß-alanine through the epidermis results in black cuticles, as has been revealed with work on mutants of various insects (33).

5. Conclusions

Many questions are open for further research. Thus, the regulatory mechanisms leading to presence of low amounts of N-acylcatecholamines in the CNS as compared with rather large amounts occurring in haemolymph and cuticle are poorly understood. Another intriguing problem to be solved concerns the fact that the oxidation of tyrosine by tyrosinase leads to DOPA but not to melanin. Much work has to be done on the various oxidative enzymes occurring in CNS, haemolymph, and cuticle. The isolation and identification of those enzymes from various organs of insects presents a formidable problem. There is always the danger of contamination of the tissues with haemolymph. Eventually, the application of tissue cultures will lead to solutions of some of the most crucial problems.

Despite many similarities in oxidative pathways of tyrosine metabolism in insects and vertebrates, some fundamental differences exist, particularly with respect to the hydroxylation of tyrosine to DOPA and the intermediacy of quinone methides in oxidation of N-acylcatecholamines. It is expected that the results of future research will add to our knowledge not only on melanin chemistry and biochemistry in general, but will also provide new aspects that are of mutual interest for the vertebrate and the invertebrate melanin scientist.

Acknowledgements: The work cited from the authors laboratory was supported by the Deutsche Forschungsgemeinschaft (Bonn), the Commission of the European Communities, Science Program (Brussels), and by the Fonds der Chemischen Industrie (Frankfurt).

References

1. Evans PD: Advan Insect Physiol, 15:317-473, 1980.
2. Brown CS, Nestler C: in Kerkut GA, Gilbert, LI (eds): Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 2, pp. 435-497 (Pergamon Press, Oxford 1985).
3. Owen MD, Bouquillon AI: Insect Biochem Mol Biol, 22:193-198, 1992.
4. Krueger RR, Kramer KJ, Hopkins TL, Speirs RD: Insect Biochem, 20:605-610, 1990.
5. Murdock LL, Wirtz RA, Köhler G: Biochem J, 132:681-688, 1973.
6. MacFarlane RG, Midgley JM, Watson DG, Evans PD: Insect Biochem, 20: 305-311, 1990.
7. Maxwell GD, Moore MM, Hildebrand JG: Insect Biochem, 10:657-665, 1980.
8. Hiruma K, Riddiford LM: Dev Biol, 138:214-224, 1990.
9. Kayser H: in Kerkut GA, Gilbert, LI (eds): Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 10, pp. 367-415 (Pergamon Press, Oxford 1985).
10. Aso Y, Kramer KJ, Hopkins, TL, Whetzel SZ: Insect Biochem, 14:463-472, 1984.
11. Binnigton U, Retnakaran A (eds): Physiology of the Insect Epidermis, CSIRO Publications, East Melbourne, 1991.
12. Hopkins TL, Kramer KJ: Ann Rev Entomol, 37:273-302, 1992.
13. Peter MG: Insect Biochem 10:221-227, 1980.
14. Peter MG, Vaupel W: J Chem Soc Chem Commun, 848-850, 1985.
15. Saul SJ, Sugumaran M: FEBS Lett, 237:155-158, 1988.
16. Saul SJ, Sugumaran M: J Biol Chem, 265:16992-16999, 1990.
17. Thomas BR, Yonekura M, Morgan TD, Czapla TH, Hopkins TL, Kramer KJ: Insect Biochem, 19:611-612, 1989.
18. Morgan TD, Thomas BR, Yonekura M, Czapla TH, Kramer KJ, Hopkins TL: Insect Biochem, 20:251-260, 1990.
19. Andersen SO, Insect Biochem, 19:375-382, 1989.
20. Saul SJ, Sugumaran M: FEBS Lett, 255:340-344, 1989.
21. Peter MG: Angew Chem Int Ed Engl, 28:555-570, 1989.
22. Schaefer J, Kramer KJ, Garrow JR, Jacob GS, Stejskal EO, Hopkins TL, Speirs RD: Science, 235:1200-1204, 1987.
23. Christensen AM, Schaefer J, Kramer KJ, Morgan TD, Hopkins TL: J Am Chem Soc, 113:6799-6802, 1991.
24. Andersen SO, Jacobsen JP, Roepstorff P, Peter MG: Tetrahedron Lett, 32:4287-4290, 1991.
25. Andersen SO, Peter MG, Poepstorff P: Insect Biochem Mol Biol (in press).
26. Andersen SO: personal communication.
27. Andersen SO, Roepstorff P: Tetrahedron, 36:3249-3252, 1980.
28. Andersen SO, Jacobsen JP, Bojesen G, Roepstorff P: Biochim Biophys Acta, 1118:134-138, 1992.
29. Miessner M, Crescenzi O, Napolitano A, Prota G, Andersen SO, Peter MG: Helv Chim Acta, 74:1205-1212, 1991.
30. Peter MG: Z. Naturforsch, 33C:912-918, 1978.
31. Peter MG, Miessner M, Hartmann R, Andersen SO, Roepstorff P: in preparation.
32. Grün L, Peter MG: Z Naturforsch, 39C:1066-1071, 1984.
33. Roseland CR, Kramer KJ, Hopkins TL: Insect Biochem, 17:21-28, 1987.