Computational Investigation of the Mechanism of Diels–Alderase PyrI4

Zou, Yike; Yang, Song; Sanders, Jacob N.; Li, Wei; Yu, Peiyuan; Wang, Hongbo; Tang, Zhijun; Li, Wen; Houk, K. N.

doi:10.1021/jacs.0c10813

Cited by 20 publications

(24 citation statements)

References 39 publications

(37 reference statements)

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“…3d) 10 . Though the intermolecular and intramolecular substrates explore different regions of the β-barrel of the PyrI4, it is intriguing how the enzyme promotes the exo-favoured DAr in both the cases 17 .…”

Section: Resultsmentioning

confidence: 99%

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Exo-selective intermolecular Diels–Alder reaction by PyrI4 and AbnU on non-natural substrates

et al. 2021

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The 100-year-old Diels–Alder reaction (DAr) is an atom economic and elegant organic chemistry transformation combining a 1,3-diene and a dienophile in a [4+2] cycloaddition leading to a set of products with several stereo centres and multiple stereoisomers. Stereoselective [4+2] cycloaddition is a challenge. Here, we describe two natural enzymes, PyrI4 and AbnU performing stereospecific intermolecular DAr on non-natural substrates. AbnU catalyses a single exo-stereoisomer by 32-fold higher than the background. PyrI4 catalyses the same stereoisomer (15-fold higher) as a major component (>50%). Structural, biochemical and fluorescence studies indicate that the dienophile enters first into the β-barrel of the enzymes followed by the 1,3-diene, yielding a stereospecific product. However, if some critical interactions are disrupted to increase the catalytic efficiency, stereoselectivity is compromised. Since it is established that natural enzymes can carry out intermolecular DAr on non-natural substrates, several hundreds of Diels-Alderases available in nature could be explored.

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Section: Resultsmentioning

confidence: 99%

“…1). It has been shown that diene and dienophile are brought together in the β-barrel in an appropriate orientation to provide the required stereochemistry 16,17 . Except for two, all natural DAses reported till date have been demonstrated to carry out intramolecular DAr and only on natural substrates identified in biosynthetic pathways 18 .…”

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confidence: 99%

Exo-selective intermolecular Diels–Alder reaction by PyrI4 and AbnU on non-natural substrates

et al. 2021

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“…5b). We carried out DFT calculations by incorporating methylamine and acetic acid as models to reproduce the hydrogen bond network (i.e., 4 -K356-E82) 22 . The modelled network provides a tight hydrogen bond on the carbonyl oxygen at the C1 of 4 (1.74 Å) due to the acidified proton of the amino group.…”

Section: Resultsmentioning

confidence: 99%

“…Although such DAases evolved independently from their own progenitors, they all exhibit high stereoselectivity and catalytic efficiency. The molecular basis of this emerging group of enzymes is gradually being revealed by genetic, biochemical and structural analyses, in combination with computational investigations 20-22 . However, the mechanisms underlying the remarkable features of naturally occurring DAases, such as the origin of stereoselectivity and their performanceas catalysts, have remained elusive.…”

Section: Introductionmentioning

confidence: 99%

Molecular basis for two stereoselective Diels-Alderases that produce decalin skeletons

Fujiyama

Kato

et al. 2021

Preprint

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Enzymes catalyzing [4+2] cycloadditions are involved in the formation of complex structures found in natural products, and play key roles in the control of stereochemistry. Fsa2 and Phm7 catalyze intramolecular [4+2] cycloaddition to form enantiomeric decalin scaffolds during the biosynthesis of HIV-1 integrase inhibitors, equisetin, and an opposite stereochemical homolog, phomasetin. Here, we solved the X-ray crystal structures of substrate-free Fsa2 and Phm7, and an inhibitor-bound Phm7 to understand the molecular basis underlying stereoselective cycloaddition reactions. Based on the crystal structures, docking simulations followed by all-atom molecular dynamics simulations provided binding models demonstrating the folding of linear polyenoyl tetramic acid substrates in the binding pocket of these enzymes, which explain the stereoselectivity in the construction of decalin scaffolds. Site-directed mutagenesis studies verified the binding models and, in combination with density functional theory calculations, clarified how hydrophilic amino acid residues in the Phm7 pocket regulate and catalyze the stereoselective cycloaddition. This powerful combination of experimental and theoretical approaches highlights the distinct molecular mechanisms of enzyme-mediated [4+2] cycloaddition and its stereoselectivity.

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“…Taking a closer look at the binding model, we discerned that the amino moiety of K356 makes another hydrogen bond with the carboxy group of E82 to form an ammonium architecture (Figure 5 b). We carried out DFT calculations by incorporating methylamine and acetic acid as models to reproduce the hydrogen bond network (i.e., 4 ‐K356‐E82) [10a] . The modelled network provides a tight hydrogen bond on the carbonyl oxygen at the C1 of 4 (1.74 Å) due to the acidified proton of the amino group.…”

Section: Resultsmentioning

confidence: 99%

Molecular Basis for Two Stereoselective Diels–Alderases that Produce Decalin Skeletons**

Fujiyama

Kato

et al. 2021

Angewandte Chemie

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Enzymes catalyzing [4+2] cycloaddition have attracted increasing attention because of their key roles in natural product biosynthesis. Here, we solved the X‐ray crystal structures of a pair of decalin synthases, Fsa2 and Phm7, that catalyze intramolecular [4+2] cycloadditions to form enantiomeric decalin scaffolds during biosynthesis of the HIV‐1 integrase inhibitor equisetin and its stereochemical opposite, phomasetin. Computational modeling, using molecular dynamics simulations as well as quantum chemical calculations, demonstrates that the reactions proceed through synergetic conformational constraints assuring transition state‐like substrates folds and their stabilization by specific protein‐substrate interactions. Site‐directed mutagenesis experiments verified the binding models. Intriguingly, the flexibility of bound substrates is largely different in two enzymes, suggesting the distinctive mechanism of dynamics regulation behind these stereoselective reactions. The proposed reaction mechanism herein deepens the basic understanding how these enzymes work but also provides a guiding principle to create artificial enzymes.

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Computational Investigation of the Mechanism of Diels–Alderase PyrI4

Cited by 20 publications

References 39 publications

Exo-selective intermolecular Diels–Alder reaction by PyrI4 and AbnU on non-natural substrates

Exo-selective intermolecular Diels–Alder reaction by PyrI4 and AbnU on non-natural substrates

Molecular basis for two stereoselective Diels-Alderases that produce decalin skeletons

Molecular Basis for Two Stereoselective Diels–Alderases that Produce Decalin Skeletons**

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