Lovastatin Nonaketide Synthase Catalyzes an Intramolecular Diels−Alder Reaction of a Substrate Analogue

Abstract

The fungal metabolite lovastatin (1) 1 and its derivatives are cholesterol-lowering drugs that act as potent inhibitors of (3S)hydroxy-3-methylglutaryl-coenzyme A reductase. 2 Although 1 and compactin 3 have attracted attention from synthetic chemists, 4 these drugs and some analogues (e.g., simvastatin, pravastatin) which are used in humans are manufactured by fermentation, either directly or with subsequent chemical or microbial modification. Studies on the biosynthesis of 1 in Aspergillus terreus indicate that it is formed by a polyketide pathway. [5][6][7] Of special interest is the proposal of an enzyme-catalyzed Diels-Alder cyclization of the intermediate hexaketide triene to generate the decalin system (Figure 1). 7,8 This idea is supported by the formation of dihydromonacolin L (2) 9 by a heterologous host, A. nidulans containing the loVB and loVC genes from A. terreus. 10,11 Corresponding heterologous expression of the loVB protein (lovastatin nonaketide synthase, LNKS) without loVC leads to truncated pyrones 3 and 4, formed due to failure of enoyl reduction at the tetraketide stage. 10 There are proposals that enzyme-catalyzed Diels Alder reactions may occur during biosynthesis of many secondary metabolites, 12 but the demonstrated ability of pure biological macromolecules to promote this process has been limited to catalytic antibodies generated from synthetic haptens 13 and to synthetic RNA fragments that bind metals. 14 There is also a report of a crude cellfree preparation from the fungus Alternaria solani that oxidizes an achiral allylic alcohol, prosolanopyrone II, to a conjugated triene aldehyde, thereby triggering intramolecular Diels-Alder cyclization to an optically active product, solanopyrone A. 15 We now report that purified LNKS catalyzes intramolecular Diels-Alder endo closure of a substrate analogue, (E,E,E)-(R)-6-methyldodecatri-2,8,10-enoic acid N-acetylcysteamine (NAC) thioester (5), to a bicyclic system with the same ring stereochemistry as 2, which is different from that obtained in nonenzymatic cyclization.

Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase

“…Although putative enzymes for unimolecular [4+2] cycloadditions have been reported (1)(2)(3)(4), no naturally occurring enzyme is known to catalyze a bimolecular Diels-Alder reaction (5,6). The generation of artificial Diels-Alderases has therefore been a longstanding and alluring goal for protein engineers.…”

Synthesis of (-)-longithorone A: Using organic synthesis to probe a proposed biosynthesis

“…Having served its purpose of controlling the atropdiastereoselectivity during the macrocyclization, the benzylic silyloxy group of 41 was removed under ionic hydrogenation conditions using trifluoroacetic acid and Et 3 SiH, affording 44 in 58% yield as a single atropisomer (Scheme 6). Removal of the TBS group generated 45 in 87% yield.…”

Exploring the Biosynthesis of Natural Products and Their Inherent Suitability for the Rational Design of Desirable Compounds through Genetic Engineering

“…One is the lovastatin nonaketide synthase (LNKS), credited with producing lovastain's decalin core structure via a DielsAlder reaction, which was originally reported by Hutchinson and Vederas et al (Scheme 1b). 6,7) Although LNKS appears capable of biosynthesizing dihydromonacolin L via an endo Diels-Alder reaction, in vitro assays revealed that such biosynthesis would require an extra enzyme, LovC. Hence, no study has unambiguously demonstrated the ability of LNKS to catalyze a Diels-Alder reaction as a single-enzymatic reaction.…”