Brassinosteroid intermediates

Some biochemical functions defined by the Arabidopsis dwarf mutants were later confirmed by heterologous expression of genes and by in vivo conversion of postulated substrates [17-20]. As part of these physiological and biochemical studies, tomato cell suspension cultures have also been established to investigate intermediates and enzymes of brassinosteroid biosynthesis and metabolism [21-23]. Enzyme activities from partially purified protein extracts were first detected in this model system [24]. 

The Arabidopsis mutant dwf7/ste 1 is defective in C5-desaturation of episterol (Fig. (2)) [18], thus impaired in an enzyme function involved in a very early step of brassinosteroid precursor biosynthesis. The enzymatic block of dwf7/stel was determined by feeding experiments using l3C-labelled mevalonic acid and a subsequent analysis of endogenous sterol and brassinosteroid precursors. The mutant accumulates episterol with a simultaneous decrease of downstream intermediates (24-methylenecholesterol, campesterol, castasterone, brassinolide). 

Another dwarf mutant of Arabidopsis, sax], defines a step upstream of DWF1 in the brassinosteroid biosynthesis pathway [27]. Rescue experiments with intermediates showed that saxl is involved in the oxidation and isomerization of 3P-hydroxyl,A5 6 precursors to 3-oxo-A4 5 steroids (Fig. (4)). 

Nevertheless, there are many questions still open because of problems to detect enzyme activities corresponding to each step of the pathway. The model of biosynthesis pathway was put together by studying the metabolism of exogenously applied intermediates in cell cultures of various origins and combining these results with data of native brassinosteroid patterns. It is more or less accepted that there are three pathways in parallel, the early and the late C6 oxidation pathway, as well as the 24/ -epimers follow ing the same route. Some observations in the analysis of native brassinosteroid patterns suggest a possible connection between the pathways. It was shown that seeds of Arabidopsis contain castasterone and 24-epi-brassinolide [34]. Also members of both 24-epimers, brassinolide and 24-epi-brassinolide were detected in tomato seeds [Winter, unpublished]. 

Suzuki, H., Inoue, T., Fujika, S., Saito, T., Takatsuko, S., Yokota, T., Murofushi, N., Yanagisawa, T. and Sakurai, A. (1995) Conversion of 24-methylcholesterol to 6-oxo-24-methylcholestanol, a putative intermediate of the biosynthesis of brassinosteroids, in cultured cells of Catharanthus roseus. Phytochemistry, 40,1391-7. 

This brief review covers the period of the last ten years in the development of brassinosteroid chemistry in our laboratory. Our interest has been focused on the synthesis of natural brassinosteroids (brassinolide, homobrassinoli-de, epibrassinolide, norbrassinolide, etc.), their analogues and intermediates starting from stigmasterol, ergosterol, or pregnenolone. Some aspects of biological activity are discussed. 

Microbial Transformation of Brassinosteroids. Until now, information concerning the microbial transformations of brassinosteroids has not been published. Such biotransformations could open novel pathways to additionally functionalized members for structure-activity investigations as well as provide information about possible metabolic processes of such compounds. Furthermore, the intermediate metabolites may have practical application. 

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