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EP-4739772-A2 - ENGINEERED ROSSMAN FOLD-CONTAINING OXIDOREDUCTASES

EP4739772A2EP 4739772 A2EP4739772 A2EP 4739772A2EP-4739772-A2

Abstract

The present invention relates to a variant of a parent NAD(P)H dependent oxidoreductase comprising (a) a Rossman fold with a consensus motif GXnGXmG/A, wherein n is 1, 2 or 3 and m is 1 or 2, and (b) a glycine residue being located 8 to 14 amino acid residues downstream of said consensus motif, wherein in the variant the glycine residue is mutated.

Inventors

  • STAUNIG, Nicole
  • DUPONT, MARIA
  • Koch, Dennis

Assignees

  • Annikki GmbH

Dates

Publication Date
20260513
Application Date
20240708

Claims (20)

  1. 1. A variant of a parent NAD(P)H dependent oxidoreductase comprising (a) a Rossman fold with a consensus motif GX n GX m G/A, wherein n is 1, 2 or 3 and m is 1 or 2, and (b) a glycine residue being located 8 to 14 amino acid residues downstream of said consensus motif, wherein in the variant the glycine residue is mutated.
  2. 2. The variant of claim 1, wherein the glycine residue is located 9 to 14, preferably 10 to 14, more preferably 10 to 13, more preferably 10 to 12, in particular 11, amino acid residues downstream of said consensus motif.
  3. 3. The variant of claim 1 or 2, wherein the glycine residue is substituted or deleted.
  4. 4. The variant of any one of claims 1 to 3, wherein the glycine residue is substituted with an amino acid residue selected from the group consisting of asparagine, arginine, cysteine, tyrosine, threonine, glutamine, leucine, alanine, aspartic acid and proline.
  5. 5. The variant of any one of claims 1 to 4, wherein n is 1 and m is 2, or n and m are 2, or n is 2 and m is 1, or n is 3 and m is 1.
  6. 6. The variant of any one of claims 1 to 5, wherein the oxidoreductase is an oxidoreductase selected from enzyme class EC 1, preferably selected from the group consisting of EC 1.1, EC 1.2, EC 1.3, EC 1.4, EC 1.5, EC 1.6, EC 1.7, EC 1.12 and EC 1.19.
  7. 7. The variant of any one of claims 1 to 6, wherein the NAD(P)H dependent oxidoreductase is selected from the group consisting of NAD(P)H oxidase, xylitol dehydrogenase, glucose 1-dehydrogenase, aldehyde dehydrogenase, hydroxysteroid dehydrogenase, lactate dehydrogenase, Glucitol (sorbitol) dehydrogenase and SDR family oxidoreductase .
  8. 8. The variant of any one of claims 1 to 7, wherein the NAD(P)H dependent oxidoreductase is an NAD(P)H oxidase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 1, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 2 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 2.
  9. 9. The variant of any one of claims 1 to 8, wherein the variant of the NAD(P)H dependent oxidoreductase, when aligned to the sequence of SEQ ID No. 1, comprises a mutation at position 170 of SEQ ID No. 1.
  10. 10. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is an xylitol dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 16, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 17.
  11. 11. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a glucose 1-dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 18, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 19.
  12. 12. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a aldehyde dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 20, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 21 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 21.
  13. 13. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a hydroxysteroid dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 22, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 23 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 23.
  14. 14. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a lactate dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 24, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 25 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 25.
  15. 15. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a Glucitol (sorbitol) dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 26, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 27 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 27.
  16. 16. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a SDR family oxidoreductase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 28, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 29 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 29.
  17. 17. A nucleic acid molecule encoding a protein according to any one of claims 1 to 16.
  18. 18. Vector comprising a nucleic acid molecule according to claim 17.
  19. 19. Host cell comprising a nucleic acid molecule according to claim 17 or a vector according to claim 18.
  20. 20. Method for obtaining a variant of a parent NAD(P)H dependent oxidoreductase having an increased enzymatic activity compared to the parent oxidoreductase comprising the steps of providing a parent oxidoreductase comprising (a) a Rossman fold with a consensus motif GX n GX m G/A, wherein n is 1, 2 or 3 and m is 1 or 2, and (b) a glycine residue being located 8 to 14 amino acid residues downstream of said consensus motif, and mutating said glycine residue.

Description

ENGINEERED ROSSMAN FOLD -CONTAINING OXIDOREDUCTASES TECHNICAL FIELD The present invention relates to the field of enzyme enginering, in particular enzymes capable of binding NAD(H) and/or NADP (H) using the Rossman fold consensus motif GXnGXmG/A. BACKGROUND ART Biocatalytic redox processes are widely used to replace expensive chemical synthesis, the processes can be performed in shorter time using milder reaction conditions (ambient pressure and temperature) and, furthermore, no extensive amounts of waste are formed . Redox reactions like the stereoselective reduction of keto groups or the regioselective oxidation of hydroxy groups are of special interest for pharma and food industries and are mostly performed by oxidoreductases , accompanied by the interconversion of the nicotinamide cofactors NAD(P)+ (oxidized form) and NAD(P)H (reduced form) . These redox reactions require a stoichiometric amount of NAD(P)+ or NAD(P)H for the conversion to be completed, however, the addition of such cofactors in industrial processes is very expensive and not cost-efficient. As cofactors and enzymes are expensive components of biocatalytic processes, enzyme engineering is essential for enhancing cost-efficiency. NAD(P) (H) -binding is achieved in many cases by the Rossman fold. The structural fingerprint of the Rossman fold is a stretch of thirty to thirty-five residues from the first two p strands and the connecting phosphate-binding helix (Bellamicini , 1996) . When the pap sequences that make up the functional 3D motif in various NAD(P) (H) -binding proteins were aligned, the initial Rossman consensus sequences G-X1-2-G-X-X-G was identified (Rossman et al., 1975; Dym and Eisenberg, 2001) . This consensus required modifications due to an alternative cofactor binding consensus (G- X1-2-G-X-X-A) in the glutathione reductase from E. coli (Scrutton et al., 1987) , and the more recently reported consensus G-X-X-X-G-I-G (SEQ ID No. 30) by Brakoulias and Jackson (2004) that ended up with the more general Rossman consensus GXnGXmG/A (n < 3, m < 2) . As the Rossman fold is found in a plethora of oxidoreductases , engineering this general cofactor-binding moiety would represent a promising and universally valid strategy to develop oxidoreductases with improved activity. Therefore, it is an object of the present invention to provide oxidoreductase variants showing an improved activity compared to the parent oxidoreductases. SUMMARY OF THE INVENTION The present invention relates to a variant of a parent NAD(P)H dependent oxidoreductase comprising (a) a Rossman fold with a consensus motif GXnGXmG/A, preferably GXnGXmG, wherein n is 1, 2 or 3 and m is 1 or 2, and (b) a glycine residue being located 8 to 14 amino acid residues downstream of said consensus motif, wherein in the variant the glycine residue is mutated. It was surprisingly found that mutating a glycine residue located 8 to 14 amino acid residues downstream of the Rossman fold consensus motif of a NAD(P)H dependent oxidoreductase show significantly increased enzymatic activity by more than 10%, preferably 20%, more preferably 30%, even more preferably 40% or more as compared to the parent NAD(P)H dependent oxidoreductase. As shown in the alignment of Fig. 1 typical Rossman fold comprising enzymes, revealed the more complex Rossman consensus motif, GXnGXmG/A (n < 3, m < 2) that harbours an additionally conserved glycine (G) residue as a secondary prototypical feature. The additionally conserved G being located 8 to 14 amino acid residues downstream of the Rossman consensus motif will be termed "secondary G" throughout the text or, when mutated, "mutated secondary G" . Thus, the present invention relates to a mutated NAD(P) (H) -dependent oxidoreductase, comprising a Rossman fold with the consensus motif G-Xn-G-Xm-G/A (n < 3, m < 2) and a secondary glycine (G) located 8 to 14 amino acids C-terminal of said consensus motif, wherein the variant is mutated at the secondary glycine (G) . A further aspect of the present invention relates to a method for obtaining a variant of a parent NAD(P)H dependent oxidoreductase having an increased enzymatic activity compared to the parent oxidoreductase comprising the steps of providing a parent oxidoreductase comprising (a) a Rossman fold with a consensus motif GXnGXmG/A, wherein n is 1 , 2 or 3 and m is 1 or 2 , and (b ) a glycine residue being located 8 to 14 amino acid residues downstream of said consensus moti f , and mutating said glycine residue . Another aspect of the present invention relates to a protein, preferably stable protein, having NADH and/or NADPH oxidase activity comprising an amino acid sequence selected from the group consisting of i ) an amino acid sequence having at least 80% sequence identity with SEQ ID No . 1 , ii ) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No . 2 of at least 80% , and iii ) an amino acid sequence encoded by a nucleic acid which binds under string