Enzymatic synthesis of carotenes and related compounds.

Data are presented in this paper which establish many of the reactions involved in the biosynthesis of carotenes. Studies have shown that all of the enzymes required for the synthesis of acyclic and cyclic carotenes from mevalonic acid are present in plastids of tomato fruits. Thus, it has been demonstrated that a soluble extract of an acetone powder of tomato fruits converts mevalonic acid to geranylgeranyl pyrophosphate, and isopentenyl pyrophosphate to phytoene, phytofluene, neurosporene and lycopene. Finally, it has been demonstrated that lycopene is converted into monoand dicyclic carotenes by soluble extracts of plastids of tomato fruits. Whether the enzymes for the conversion of acetyl-CoA to mevalonic acid are also present in tomato fruit plastids has not yet been determined. INTRODUCTION Studies on the enzymatic synthesis of carotenes were, until very recently, plagued by a number of problems. One of these was the fact that the enzymes for the synthesis of carotenes are located in a particulate body, namely chromoplasts or chloroplasts. Hence, a method of solubilization of the enzymes without appreciable loss of enzyme activity was needed. A second problem was concerned with the commercial unavailability of labelled substrate other than mevalonic acid. Thus it became necessary to synthesize other substrates either chemically or enzymatically and then to purify these compounds. Thirdly, the reactions in the synthesis of carotenes appear to proceed much more slowly than many other biochemical reactions. It is probable that this problem is not primarily one of activity of the enzyme, but instead is due to the presence of a relatively small amount of the enzyme in question in the cell. Finally, the hydrocarbon nature of the carotenes required the development of a system in which the hydrophobic substrate or intermediate could be kept in contact with the enzyme. Fortunately, most of the difficulties mentioned above have been overcome. As a result, it is now possible to discuss carotene biosynthesis in terms of demonstrable reactions, cofactor requirements, and to some extent properties of enzymes. Thus, as will be shown in this paper, the main outlines of the pathway of carotene biosynthesis have been established. However, in spite of this progress, many questions concerning specific reactions, mechanisms 449 JOHN W. PORTER of reactions, and the properties of the proteins catalyzing the synthesis of carotenes remain to be answered in future symposia. In this paper data are presented on the pathway of carotene biosynthesis and on some of the individual reactions of this pathway. Brief mention is also made of the characteristics of some of the enzymes. Most of the studies reported are concerned with enzymes obtained from tomato fruits. Other studies have utilized rat, pigeon and pig liver enzyme systems. Most of the information that is presented has been taken from studies performed in the author's laboratory. FORMATION OF C20 INTERMEDIATE Conversion of acetate to mevalonic acid Acetate is considered as the starting compound in the biosynthesis of carotenes. This compound, and its coenzyme-A derivative, is converted through a series of reactions, to the various carotenes that are found in tomato fruits. Relatively few investigations have been made on the conversion by plant enzymes of acetyl-CoA to intermediates in carotene biosynthesis. Hence, most of the information that will be considered on the earlier reactions in carotene biosynthesis will be drawn from experiments with .0 H3C—g_.bH Mg 0 GSH, CoASH