Enzyme catalysts for diels-alder reactions

Abstract

The present invention provides enzyme catalysts for Diels-Alder reactions, including intermolecular Diels-Alder reactions, as well as protein scaffolds for making such enzyme catalysts. In other aspects, the invention provides methods of making the enzyme catalysts, including by de novo computational design. The present invention thereby provides enzyme catalysts capable of catalyzing a desired Diels-Alder reaction, including with a specified or desired stereo-selectivity.

1 . An non-naturally occurring enzyme catalyst of a Diels-Alder reaction, the enzyme comprising a non-immunoglobulin scaffold, and an active site pocket that accommodates a Diels-Alder reaction. 
     
     
         2 . The enzyme of  claim 1 , wherein the enzyme comprises a protein scaffold corresponding to diisopropylfluorophosphosphatase from  Loligo vulgaris , or a homolog thereof, or ketosteroid isomerase from  Pseudomonas putida , or a homolog thereof. 
     
     
         3 . The enzyme of  claim 2 , wherein the active site comprises amino acid side chains that stabilize the Diels-Alder transition state by electron-withdrawing and/or electron-donating effects. 
     
     
         4 . The enzyme of  claim 3 , wherein the active site comprises an amino acid side chain that stabilizes the Diels-Alder transition state by accepting a hydrogen bond from the diene portion of the transition state. 
     
     
         5 . The enzyme of  claim 3 , wherein the active site comprises an amino acid side chain that stabilizes the Diels-Alder transition state by donating a hydrogen bond to the dienophile portion of the transition state. 
     
     
         6 . The enzyme of  claim 1 , wherein the enzyme contains a substrate-binding pocket having a hydrophobic and/or polar and/or charged interface that is complementary to the Diels-Alder substrates. 
     
     
         7 . The enzyme of  claim 4 , wherein the hydrogen-bond acceptor at the position corresponding to position 195 of SEQ ID NO:2. 
     
     
         8 . The enzyme of  claim 7 , wherein the hydrogen bond acceptor is a side chain of asparagine or glutamine. 
     
     
         9 . The enzyme of  claim 5 , wherein the hydrogen-bond donor is at the position corresponding to position 121 of SEQ ID NO:2. 
     
     
         10 . The enzyme of  claim 9 , wherein the hydrogen bond donor is the side chain of tyrosine, threonine, or serine. 
     
     
         11 . The enzyme of  claim 1 , wherein the amino acid sequence of the enzyme has from about 5 to about 30 amino acid substitutions with respect to SEQ ID NO:2 or 9. 
     
     
         12 . The enzyme of  claim 1 , wherein the amino acid sequence contains one or more amino acid insertions and/or deletions with respect to SEQ ID NO:2 or 9. 
     
     
         13 . The enzyme of  claim 1 , wherein the active site pocket is lined substantially with non-polar amino acids. 
     
     
         14 . The enzyme of  claim 11 , wherein one or more mutations are at the positions corresponding to positions 21, 36, 37, 39, 72, 74, 90, 120, 135, 136, 144, 146, 148, 149, 173, 175, 176, 196, 225, 229, 230, 244, 269, 271, 272 and 287 of SEQ ID NO:2. 
     
     
         15 . The enzyme of  claim 11 , wherein at least 5 mutations are at the positions corresponding to positions 21, 36, 37, 39, 72, 74, 90, 120, 135, 136, 144, 146, 148, 149, 173, 175, 176, 196, 225, 229, 230, 244, 269, 271, 272 and 287 of SEQ ID NO:2. 
     
     
         16 . The enzyme of  claim 11 , wherein at least 10 mutations are at the positions corresponding to positions 21, 36, 37, 39, 72, 74, 90, 120, 135, 136, 144, 146, 148, 149, 173, 175, 176, 196, 225, 229, 230, 244, 269, 271, 272 and 287 of SEQ ID NO:2. 
     
     
         17 . The enzyme of  claim 11 , wherein the amino acid sequence of the enzyme has at least 20% identity to the amino acid sequence of SEQ ID NO:2 or 9. 
     
     
         18 . The enzyme of  claim 1 , wherein the enzyme has the amino acid sequence of SEQ ID NO:4, optionally with from 1 to about 10 amino acid substitutions, insertions, and/or deletions with respect to SEQ ID NO:4. 
     
     
         19 . The enzyme of  claim 1 , wherein the enzyme has the amino acid sequence of SEQ ID NO:6, optionally with from 1 to about 10 amino acid substitutions, insertions, and/or deletions with respect to SEQ ID NO:6. 
     
     
         20 . The enzyme of  claim 1 , wherein the enzyme has the amino acid sequence of SEQ ID NO:8, optionally with from 1 to about 10 amino acid substitutions, insertions, and/or deletions with respect to SEQ ID NO:8. 
     
     
         21 . The enzyme of  claim 1 , wherein the enzyme catalyzes a Diels-Alder reaction involving a diene having a substituent defined by —N(H)R, or a substituent allowing for a substantially similar hydrogen-bonding geometry. 
     
     
         22 . The enzyme of  claim 1 , wherein the enzyme catalyzes a Diels-Alder reaction involving a dienophile having a substituent defined by —C(O)R, or a substituent with a substantially similar hydrogen-bonding geometry. 
     
     
         23 . The enzyme of  claim 1 , wherein the enzyme catalyzes a stereo-selective Diels-Alder reaction. 
     
     
         24 - 48 . (canceled)