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

Abstract

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.

Enzymatic intermolecular Diels-Alder reactions in synthesis: From nature to design

“…Despite significant achievements in this field, naturally-occurring intermolecular Diels-Alderases are still very limited compared with intramolecular Diels-Alderases. Recently, two groups have individually reported that intramolecular Diels-Alderases (TbtD [90], Pylr4 [91]) could also perform stereospecific intermolecular D-A reaction on non-natural substrates, opening a new window for discovering more new-to-nature enzymatic intermolecular D-A reactions from the reported intramolecular Diels-Alderases via substrate engineering or protein engineering.…”

Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

“…Interestingly, these two families of enzymes differ significantly in both their molecular architectures and catalytic mechanisms. [5b,7] To date, five enzymes catalysing spirotetronate formation have been characterised in detail, PyrI4, [7] AbyU, [8] AbmU, [9] AbnU [10] and PloI4. [11] Intriguingly, the spirotetronate cyclases exhibit modest primary amino acid sequence identity (< 25 %; Tables S2 and S3), but retain a common overall fold.…”

“…Interestingly, these two families of enzymes differ significantly in both their molecular architectures and catalytic mechanisms. [ 5b , 7 ] To date, five enzymes catalysing spirotetronate formation have been characterised in detail, PyrI4, [7] AbyU, [8] AbmU, [9] AbnU [10] and PloI4. [11] …”

Improvement of multicatalytic properties of nitrilase from Paraburkholderia graminis for efficient biosynthesis of 2‐chloronicotinic acid

“…The existence of by‐product amides during carboxylic acids biosynthesis not only reduces the yield of target product but also results in complicated purification process (Zhang et al, 2019). In addition, when catalyzing nonnatural substrates, enzymes are often subject to bottlenecks of low catalytic efficiency, poor regio‐, stereo‐, and reaction specificity (Engelskirchen et al, 2021; Kashyap et al, 2021; Reipa et al, 2002; Schroven et al, 2007). Therefore, engineering of nitrilases to expand their applications has become the research hotspot in recent years.…”