Recent Advances in Enzymatic Complexity Generation: Cyclization Reactions

Abstract

Enzymes in biosynthetic pathways, especially in plant and microbial metabolism, generate structural and functional group complexity in small molecules by conversion of acyclic frameworks to cyclic scaffolds via short, efficient routes. The distinct chemical logic used by several distinct classes of cyclases, oxidative and non-oxidative, has recently been elucidated by genome mining, heterologous expression, and genetic and mechanistic analyses. These include enzymes performing pericyclic transformations, pyran synthases, tandem acting epoxygenases, and epoxide "hydrolases", as well as oxygenases and radical S-adenosylmethionine enzymes that involve rearrangements of substrate radicals under aerobic or anaerobic conditions.

Figure 1

Three classical pericyclic reactions in Enzyme Systems. (A) Claisen rearrangement, Cope rearrangement, and [4+2] cyclizations; (B) Claisen rearrangement catalyzed by chorismate mutase; (C) reverse O-prenyl-Tyr to normal C-prenyl-Tyr is a nonenzymatic epiphenomenal Claisen rearrangement during cyanobactin assembly; (D) proposed Cope rearrangement in enzymatic formation of 4-dimethylallyl tryptophan (4-DMAT) catalyzed by 4-DMAT synthase.

Figure 2

Cope rearrangement during cyanobacterial indole monoterpene biosynthesis. (A) Structures of hapalindole, fischerindole, ambiguine, and welwitindoles; (B) formation of 3-geranyl-3-isocyanovinyl-indoleinene by geranyltransferase action; followed by Cope rearrangement and aza-Prins reaction; a proposed three way partition to product families.

Figure 3

Examples of enzyme catalyzed [4+2] cycloadditions. (A) Cyclizations catalyzed by LovB, SpnF, and AbyU; (B) Tandem [4+2] enzymatic rearrangements in pyrroindomycin pathway: decalin formation (PyrE3) followed by spirotetronate formation (Pyrl4)

Figure 4

Proposed Aza [4+2] cycloaddition reaction to form the pyridine core of thiazolyl peptides, while simultaneously creating the peptide macrocycle of this antibiotic class.

Figure 5

Reactions Catalyzed by LepI in leporin C biosynthesis, including intramolecular hetero Diels-Alder route to leporin C; a competing Diels-Alder cyclization, which can be followed by retro-Claisen rearrangement to leporin C.

Figure 6

Action of 3-prenyl-4-hydroxybenzoate decarboxylase and ferulate decarboxylase. Formation of tricyclic prenylated FAD adduct as active form of coenzyme and proposed 1,3-dipolar addition/elimination route for decarboxylation.

Figure 7

Reductive route to the tricyclic 5-6-5 framework in ikarugamycin and related polycyclic tetramate macrocycles. NADPH-mediated reductive cyclizations form the cyclopentanes with an interspersed [4+2] cyclohexene-forming reaction.

Figure 8

Tetrahydropyran (THP) formation from oxa-conjugate additions. (A) Pyran synthase catalyzed THP formation in pederin pathway; (B) DH catalyzed tandem dehydration and cyclization in ambruticin biosynthesis; (C) SalBIII catalyzed tandem dehydration and cyclization in salinomycin biosynthesis.

Figure 9

Hydroalkoxylation catalyzed by PhnF in herqueinone biosynthesis.

Figure 10

β-Lactone and β-Lactam ring biosynthesis. (A) Penicillin, cephalosporin and carbapenem core structures; (B) lipstatin and obafluorin structures; (C) tandem action of OleC and OleB generates cis-β-lactones as intermediates to cis-olefins; (D) lactonizing release of obafluorin from NRPS thioesterase domain; (E) ATP-dependent nonoxidative route for cyclization of carboxyethyl arginine into β-lactam by β-lactam synthase.

Figure 11

Tandem actions of epoxygenases and epoxide cleaving partner enzymes. (A) Squalene epoxidase and oxidosqualene cyclase; (B) Lsd18 and Lsd19 act in tandem to epoxidize two olefins and then convert them to furan and pyran heterocycles in lasalocid maturation; (C) Formation of the bicyclooctane framework of aurovertin A involves three “disappearing” epoxide rings.

Figure 12

Two additional flavoenzyme epoxidations in fungal quinolone alkaloid biosynthesis. (A) Epoxidation in the penigequinolone pathway is followed by a Brønsted acid catalyzed epoxide rearrangement; (B) Epoxidation in the aspoquinolone pathway precedes rearrangement to the fused 3–5 ring system of the final aspoquinolone framework.

Figure 13

Diversion of intermediate radicals to non-oxygenated products in iron-”oxygenase” catalysis. (A) The canonical OH• rebound mechanism to substrate S•; (B) OxyABCE actions in teicoplanin biosynthesis builds crosslinked side chains via phenoxyradicals. All the phenyl coupling steps take place on the peptidyl carrier protein (PCP) before release by action of a thioesterase domain; (C) proposed catalytic mechanism of OxyB that does not include OH• rebound.

Figure 14

Isopenicillin N synthase (IPNS) and desacetoxycephalosporin C synthase (DAOCS) reduce O₂ but do not incorporate oxygen atoms into the 4,5-ring system of penicillin or the 4,6-expaneded bicyclic system of cephems.

Figure 15

Oxidative cyclization steps catalyzed by oxygenases that use high valent oxo-iron species in the biosynthesis of okaramine E.

Figure 16

Two distinct modes of reactivity of S-adenosylmethionine. Top, two electron reactivity, as donor of [CH₃⁺] equivalents to cellular nucleophiles; bottom, one electron reactivity as SAM is cleaved to the 5′-deoxyadenosyl radical (5′-dA•) as initiator of substrate radicals by H• removal from bound substrate.

Figure 17

Radical SAM enzymes act via 5′-deoxyadenosyl radicals to generate substrate radicals. (A) PQQ is carved out of a protein precursor with C-C bond formation between Glu and Tyr residues via radical chemistry; (B) Contraction of the ribose ring in 2′-deoxy-AMP to the oxetane aldehyde during oxetanocin A biosynthesis involves both 5′-dA• as radical initiator and the Co^II form of B12 as radical terminator.

Figure 18

Three modes of SAM fragmentation in tricyclic wybutosine assembly in tRNA. Three SAMs donate [CH₃⁺] equivalents; one SAM donates an [aminobutyryl⁺] equivalent. Two SAMs are involved in the donation of a [CH₃•] equivalent^, one is cleaved to SAH while the other is cleaved to methionine and 5′-deoxyadenosine.