Control of the Stereochemical Course of [4+2] Cycloaddition during trans‐Decalin Formation by Fsa2‐Family Enzymes

Abstract

Enzyme-catalyzed [4+ +2] cycloaddition has been proposed to be akey transformation process in various natural product biosynthetic pathways. Recently Fsa2 was found to be involved in stereospecific trans-decalin formation during the biosynthesis of equisetin, apotent HIV-1 integrase inhibitor.T o understand the mechanisms by which fsa2 determines the stereochemistry of reaction products,w es ought an fsa2 homologue that is involved in trans-decalin formation in the biosynthetic pathway of an enantiomerically opposite analogue,a nd we found phm7, which is involved in the biosynthesis of phomasetin. Ad ecalin skeleton with an unnatural configuration was successfully constructed by gene replacement of phm7 with fsa2, thus demonstrating enzymatic control of all stereochemistry in the [4+ +2] cycloaddition. Our findings highlight enzyme-catalyzed [4+ +2] cycloaddition as as tereochemically divergent step in natural product biosynthetic pathwaysa nd open new avenues for generating derivatives with different stereochemistry.

Predicting the chemical space of fungal polyketides by phylogeny-based bioinformatics analysis of polyketide synthase-nonribosomal peptide synthetase and its modification enzymes

“…3 and S2 , Table S6 ). Additionally, we found that both type-I and type-II DAases can be further divided into two subclades: clade Iα functionally characterized DAase: LepI 25 ), Iβ (IccD 23 ), IIα (EqxF 31 , Fsa2 32 , Phm7 32 , and Tas3 33 ), and IIβ (CcsF 34 , CHGG_01241 35 , PoxQ 36 , and MycB 37 ). IIβ can be further divided into two groups, which reflect the two alternative functions of synthesizing either a macrocyclic (IIβ-1) or decalin (IIβ-2) ring.…”

Enzyme evolution in fungal indole alkaloid biosynthesis

“…This was due to the unique identities of the respective Diels-Alderases that result from divergent evolutionary processes. Recent studies have shown that Diels-Alderase enzymes typically lose their original function and have evolved to control stereoselectivity for the cycloaddition reaction [22,[40][41][42][43][44][45][46]. Additionally, bound cofactors no longer serve a catalytic function (e.g., methyl transfer, redox), and only play a structural role in maintaining the proper active site conformation for stereochemical control [40][41][42][43][44][45][46].…”

“…Recent studies have shown that Diels-Alderase enzymes typically lose their original function and have evolved to control stereoselectivity for the cycloaddition reaction [22,[40][41][42][43][44][45][46]. Additionally, bound cofactors no longer serve a catalytic function (e.g., methyl transfer, redox), and only play a structural role in maintaining the proper active site conformation for stereochemical control [40][41][42][43][44][45][46]. Moreover, most Diels-Alderases have no identified catalytic residues, leading to the conclusion that these biocatalysts function by constraining the substrate in the proper orientation [40][41][42][43][44][45][46].…”

“…Additionally, bound cofactors no longer serve a catalytic function (e.g., methyl transfer, redox), and only play a structural role in maintaining the proper active site conformation for stereochemical control [40][41][42][43][44][45][46]. Moreover, most Diels-Alderases have no identified catalytic residues, leading to the conclusion that these biocatalysts function by constraining the substrate in the proper orientation [40][41][42][43][44][45][46]. Within this class of bicyclo[2.2.2]diazaoctane ring-containing molecules, the cycloaddition is proposed to be stereospecific based on the synor anti-configuration of the bridged bicycle relative to C6 ( Fig.…”

An Interpreted Atlas of Biosynthetic Gene Clusters from 1000 Fungal Genomes

“…The equisetin biosynthetic pathway involves three major steps: assembly of a decalin core via the action of polyketide synthase (PKS) enzyme domains and a Diels Alderase, formation of an amino acid-derived tetramic acid moiety catalyzed by NRPS domains, and N-methylation of the tetramic acid moiety (Fig. S12) (37,39). While the domain structure of the PKS contained in the equisetin GCF remains consistent across fungi, differences in backbone enzyme amino acid sequence and the presence/absence of tailoring enzymes mediate structural variations to the scaffold.…”