Phosphodeoxyribosyl transferase mutant enzymes and uses thereof

Patent metadata

Field	Value
Publication number	US-9523084-B2
Application number	US-201314441560-A
Country	US
Kind code	B2
Filing date	Nov 8, 2013
Priority date	Nov 8, 2012
Publication date	Dec 20, 2016
Grant date	Dec 20, 2016

Abstract

The present invention relates to enzyme mutants obtained from the nucleoside deoxyribosyltransferase (NDT) of Lactobacilli bacteria. These mutants are surprisingly able to exchange deoxyribose mono-, bi- and triphosphate between various nucleobases. In vitro syntheses have been successfully performed with several nucleobase analogs. These enzyme mutants provide new tools to synthesize, in vitro and/or in vivo, dNTPs or analogs thereof.

First claim

The invention claimed is:

1. A mutant phosphodeoxyribosyltransferase enzyme comprising amino acid sequence SEQ ID NO:1, in which the Aspartic acid at position 92 is replaced by a Serine (S) or a Glycine (G), provided that, when the Aspartic acid at position 92 is replaced by a Glycine (G), the Valine amino acid at position 93 is replaced by a Threonine (T), or a Proline (P), or a Serine (S) or a Glycine (G).

2. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:3 corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Serine (S).

3. The mutant enzyme of claim 1 , further comprising at least one mutation selected from the group consisting of:
(1) the Asparagine amino acid at position 123 is replaced by Serine (S);
(2) the Phenylalanine amino acid at position 13 is replaced by an Arginine (R) or a Glutamine (Q);
(3) the Glutamic acid at position 91 is replaced by a Glutamine (Q); and
(4) the Tyrosine amino acid at position 157 is replaced by a Lysine (K) or an Arginine (R).

4. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:4, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Serine (S) and the amino acid at position 123 is replaced by a Serine (S).

5. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:5, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Serine (S) and the amino acid at position 13 is replaced by an Arginine (R).

6. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:6, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Serine (S), the amino acid at position 13 is replaced by an Arginine (R), and the amino acid at position 123 is replaced by a Serine (S).

7. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:7, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Serine (S), the amino acid at position 13 is replaced by a Glutamine (Q), and the amino acid at position 123 is replaced by a Serine (S).

8. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:8, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by an Arginine (R), the amino acid at position 123 is replaced by a Serine (S), and the amino acid at position 93 is replaced by a Threonine (T).

9. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:9 corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by an Arginine (R), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 93 is replaced by a Threonine (T) and the amino acid at position 157 is replaced by a Lysine (K).

10. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:10, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by an Arginine (R), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 93 is replaced by a Threonine (T) and the amino acid at position 91 is replaced by a Glutamine (Q).

11. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:35, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by a Glutamine (Q), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 93 is replaced by a Threonine (T) and the amino acid at position 157 is replaced by a Lysine (K).

12. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:36, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Serine (S), the amino acid at position 13 is replaced by a Glutamine (Q), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 91 is replaced by a Glutamine (Q) and the amino acid at position 157 is replaced by a Lysine (K).

13. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:37, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by a Glutamine (Q), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 93 is replaced by a Threonine (T), the amino acid at position 91 is replaced by a Glutamine (Q) and the amino acid at position 157 is replaced by a Lysine (K).

14. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:38, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by an Arginine (R), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 93 is replaced by a Proline (P), the amino acid at position 91 is replaced by a Glutamine (Q,) and the amino acid at position 157 is replaced by a Lysine (K).

15. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:39, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by an Arginine (R), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 93 is replaced by a Glycine (G), the amino acid at position 91 is replaced by a Glutamine (Q,) and the amino acid at position 157 is replaced by a Lysine (K).

16. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:40, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by an Arginine (R), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 93 is replaced by a Serine (S), the amino acid at position 91 is replaced by a Glutamine (Q,) and the amino acid at position 157 is replaced by a Lysine (K).

17. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:41, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by an Arginine (R), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 93 is replaced by a Proline (P), the amino acid at position 91 is replaced by a Glutamine (Q,) and the amino acid at position 157 is replaced by an Arginine (R).

18. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:42, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by an Arginine (R), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 93 is replaced by a Glycine (G), the amino acid at position 91 is replaced by a Glutamine (Q,) and the amino acid at position 157 is replaced by an Arginine (R).

19. The mutant enzyme of claim 1 comprising amino acid sequence SEQ ID NO:43, corresponding to SEQ ID NO:1 in which the amino acid at position 92 is replaced by a Glycine (G), the amino acid at position 13 is replaced by an Arginine (R), the amino acid at position 123 is replaced by a Serine (S), the amino acid at position 93 is replaced by a Serine (S), the amino acid at position 91 is replaced by a Glutamine (Q) and the amino acid at position 157 is replaced by an Arginine (R).

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Assignees

• Centre Nat Rech Scient
• Centre Nat De La Rech Scient (Cnrs)

Inventors

• Kaminski Pierre-Alexandre
• Labesse Gilles

Classifications

• C12N9/1077 Primary

  Pentosyltransferases (2.4.2) · CPC title

• C12Y204/02006

  Nucleoside deoxyribosyltransferase (2.4.2.6) · CPC title

• C12N9/2497

  hydrolysing N- glycosyl compounds (3.2.2) · CPC title