Biosynthesis of Polyketides

BY T. J. SIMPSON

1 Introduction

This chapter follows the format of the previous report with the inclusion of an extra section on compounds of mixed polyketide–terpenoid origin, and covers the literature from late 1976 until the end of 1978. 13C-Labelling studies now greatly outnumber those using 14C, although 13C-methods are still almost exclusively limited to studies with micro-organisms. An interesting and potentially very useful development has been in the use of 2H-labelling, the label being detected either directly by 2H n.m.r., or indirectly through its coupling to 13C in the 13C n.m.r. spectra of metabolites enriched from doubly labelled [2H, 13C] precursors. These methods may be of even more use in the study of terpenoid biosynthesis. 3H-Labelling in conjunction with 3H n.m.r. should also be of use, but it has been applied in only one major study to date. There has been an encouraging number of papers describing the incorporation of larger molecules to obtain details of the later stages of polyketide biosynthesis; and close examination of the fate of 13C-label through secondary incorporation routes has shed light on the overall metabolic pathways operating in many organisms. An excellent short review of the fundamentals of polyketide biosynthesis has appeared.

2 Fatty Acids, Polyacetylenes, and Prostaglandins

In a series of related papers, from Cornforth and co-workers, the precise stereochemical course of individual reactions in the biosynthesis of fatty acids was investigated. Incubation of chiral acetates with either chicken-liver or bakers' yeast fatty-acid synthetase showed that in palmitic acid a higher proportion of tritium was retained from (S)-[2-14C, 3H1]acetyl-CoA than from the (R) isomer, indicating an overall stereospecificity in the formation of fatty acids from chiral acetate. The discrimination between the (R) and (S) isomers was small because the small hydrogen isotope effect, operative in the acetyl-CoA carboxylase reaction, led to nearly equal proportions of (R)- and (S)-tritiated malonyl-CoA species. A partial and non-specific exchange of hydrogen catalysed by the synthetase was also observed, and this further reduced the net retention of tritium. Malonyl thiol esters stereospecifically labelled with tritium at C-2 were prepared, and incubation of these chiral malonates with the purified yeast synthetase showed that (2S)-malonate retained 51% of the original tritium whereas the (2R)-isomer retained only 23%. Comparison of these results with those from chiral acetates indicates that carboxylation of acetyl-CoA occurs with retention of configuration. It is known that reduction of the 3-ketoacyl product (1) from condensation of acetyl-CoA with malonyl-CoA is stereospecific, giving rise to the (3R)-hydroxyacyl intermediate (2) which in turn dehydrates to give exclusively the trans-2-enoyl derivative (3). Dehydration of (2R,3R)-3-hydroxy[2-3H1]butyryl thioester by yeast fatty acid synthetase proceeded with retention of tritium to give the trans-2-enoyl derivative, by means of a syn elimination of the elements of water. Assignment of the syn stereochemistry for the dehydration, together with the findings that tritium is retained preferentially from (2S)-[2-3H1]malonate, means that the condensation reaction in fatty acid biosynthesis must proceed with inversion of configuration at C-2 of malonate. Thus the overall stereochemistry of the process is as shown in Scheme 1.

Lynen has examined the selective inhibition of fatty acid synthetase to obtain information on the condensation step in fatty acid biosynthesis. The synthetase reacts with three moles of iodoacetamide, resulting in inhibition of condensation activity, but greatly increasing the malonyl-CoA decarboxylase activity which is normally low. To account for this, a mechanism is proposed (Scheme 2) in which binding of iodoacetamide to the peripheral thiol groups induces the same change in enzyme conformation as does acetyl-CoA, permitting binding of malonate to the active site as usual. Now, however, instead of concerted loss of CO2 and transfer of malonate to the acyl residue [path (a)], simple decarboxylation results as shown in path (b). Further reaction of the inhibited synthetase with three moles of N-ethylmaleimide results in inhibition of the decarboxylase activity also.

The biosynthesis of cyclopentenyl fatty acids from 2-cyclopentenyl carboxylic (aleprolic) acid (4) was tested in seeds and leaves of the Flacourtiaceae viz. Caloncoba echinata and Hydnocarpus anthelminthica, and in various preparations of other higher plants. Only tissues of the Flacourtiacae, where the cyclopentenyl fatty acids occur naturally, were able to accept aleprolic acid as a starter for fatty acid synthesis. The labelling pattern of both straight chain and cyclic fatty acids synthesized after incubation of Flacourtiaceae seeds with [1-14C]acetate indicated initial de novo synthesis of C16 fatty acids in each case, followed by chain elongation to higher homologues. Cyclopentenylglycine, which is found in the tissues of Flacourtiaceae, where it is believed to be formed from acetate and glutamate, serves as a precursor for the cyclic fatty acids, transamination and decarboxylation converting it to aleprolic acid.

Multibranched fatty acids, e.g. 2,4,6,8-tetramethyldecanoic acid (5), are the major fatty acids produced by goose uropygial gland. The enzyme systems from this gland and from goose liver have both been shown to be equally capable of producing both branched and non-branched fatty acids, so that the production of methyl substituted fatty acids is not an inherent property of the enzyme system but simply reflects the availability of methylmalonyl-CoA in the uropygial gland. Radiclonic acid (6) is a multi-branched fatty acid produced by a Pencillium sp. Incorporation of [13C]acetate and methionine confirmed its biosynthesis by the usual fungal pathway via methylation of an intermediate polyketide chain and not from propionate.

Incorporation of octanoic acids tritiated at C-5, C-6, C-7, and C-8 into lipoic acid (7) by cultures of E. coli indicated complete tritium retention at C-5, C-7, and C-8 thus excluding unsaturated intermediates. Fifty per cent of the tritium at C-6, however, was lost, and a further experiment with (6R)- and (6S)-[6-3H1]-octanoate indicated that sulphur was introduced at C-6 with loss of the 6-pro-R hydrogen and inversion of configuration (Scheme 3).

Polyacetylenes with eight carbon atoms are produced exclusively in fungal cultures. Feedings of [18-14C]crepenynate (8),...