Method for producing a deuterated or tritiated NAD(P)H

Patent metadata

Field	Value
Publication number	US-12545940-B2
Application number	US-201916969007-A
Country	US
Kind code	B2
Filing date	Feb 12, 2019
Priority date	Feb 12, 2018
Publication date	Feb 10, 2026
Grant date	Feb 10, 2026

Abstract

Described herein is a method of producing a reduced labelled cofactor comprising one or more x H atom, wherein x is 2 or 3. Described herein is also a method of producing a reduced labelled reaction product comprising one or more x H atom, wherein x is 2 or 3, wherein the method comprises producing a reduced labelled cofactor according to the invention. Described herein are also systems for performing such methods.

First claim

The invention claimed is:

1 . A method of producing a reduced labelled cofactor comprising one or more x H atoms, wherein x is 2 or 3, wherein said method comprises:
(i) providing a composition comprising x H + ions and an oxidised cofactor; (ii) transferring electrons from an electron source to a first polypeptide, which is an NADH:acceptor oxidoreductase or an NADPH:acceptor oxidoreductase; and (iii) contacting the x H + ions and the oxidised cofactor with the first polypeptide, thereby reducing the oxidised cofactor to form a reduced labelled cofactor comprising one or more x H + atoms.

2 . The method according to claim 1 , wherein the x H + ions are provided as 2 H 2 O or 3 H 2 O.

3 . The method according to claim 1 , wherein the oxidised cofactor is NAD + , NADP + , or a labelled version of NAD + or NADP + comprising at least one x H atom.

4 . The method according to claim 1 , wherein the x H + ions are transferred directly from the composition comprising x H + ions to the first polypeptide.

5 . The method according to claim 1 , wherein the first polypeptide transfers an x H − anion to the oxidised cofactor, thereby forming the reduced labelled cofactor.

6 . The method according to claim 1 , wherein:
a) the first polypeptide comprises a flavin group; b) the first polypeptide has a structure comprising a Rossmann fold; and/or c) the first polypeptide comprises or consists of a diaphorase moiety.

7 . The method according to claim 1 , wherein the first polypeptide comprises or consists of one or more of:
i) an amino acid sequence of Ralstonia eutropha diaphorase HoxF comprising SEQ ID NO: 1, or an amino acid sequence having at least 99% identity therewith; ii) an amino acid sequence of Ralstonia eutropha diaphorase HoxU comprising SEQ ID NO: 2, or an amino acid sequence having at least 99% identity therewith; iii) an amino acid sequence of Ralstonia eutropha diaphorase HoxI comprising SEQ ID NO: 3, or an amino acid sequence having at least 99% identity therewith; iv) an amino acid sequence of the 51 kDa protein of the flavoprotein (Fp) subcomplex of Complex I of Bos taurus comprising SEQ ID NO: 4, or an amino acid sequence having at least 99% identity therewith; v) an amino acid sequence of the 24 kDa subcomplex of Complex I of Bos taurus comprising SEQ ID NO: 5, or an amino acid sequence having at least 99% identity therewith; vi) an amino acid sequence of Ralstonia eutropha NAD + -dependent formate dehydrogenase diaphorase moiety FdsB comprising SEQ ID NO: 6, or an amino acid sequence having at least 99% identity therewith; vii) an amino acid sequence of Ralstonia eutropha NAD + -dependent formate dehydrogenase diaphorase moiety FdsG comprising SEQ ID NO: 7, or an amino acid sequence having at least 99% identity therewith; viii) an amino acid sequence of Rhodobacter capsulatus NAD + -dependent formate dehydrogenase diaphorase moiety FdsB comprising SEQ ID NO: 8, or an amino acid sequence having at least 99% identity therewith; ix) an amino acid sequence of Rhodobacter capsulatus NAD + -dependent formate dehydrogenase diaphorase moiety FdsG comprising SEQ ID NO: 9, or an amino acid sequence having at least 99% identity therewith; x) an amino acid sequence of the NADPH oxidoreductase moiety from Pyrococcus furiosus soluble hydrogenase I gamma subunit comprising SEQ ID NO: 10, or an amino acid sequence having at least 99% identity therewith; xi) an amino acid sequence of the NADPH oxidoreductase moiety from Pyrococcus furiosus soluble hydrogenase I beta subunit comprising SEQ ID NO: 11, or an amino acid sequence having at least 99% identity therewith; xii) an amino acid sequence of the NADPH oxidoreductase moiety from Pyrococcus furiosus soluble hydrogenase II gamma subunit comprising SEQ ID NO: 12, or an amino acid sequence having at least 99% identity therewith; xiii) an amino acid sequence of the amino acid sequence of the NADPH oxidoreductase moiety from Pyrococcus furiosus soluble hydrogenase II beta subunit comprising SEQ ID NO: 13, or an amino acid sequence having at least 99% identity therewith; xiv) an amino acid sequence of the diaphorase moiety of Rhodococcus opacus soluble hydrogenase HoxF comprising SEQ ID NO: 14, or an amino acid sequence having at least 99% identity therewith; xv) an amino acid sequence of the diaphorase moiety of Rhodococcus opacus soluble hydrogenase HoxU comprising SEQ ID NO: 15, or an amino acid sequence having at least 99% identity therewith; xvi) an amino acid sequence of the diaphorase moiety of Allochromatium vinosum soluble hydrogenase HoxF comprising SEQ ID NO: 16, or an amino acid sequence having at least 99% identity therewith; xvii) an amino acid sequence of the diaphorase moiety of Allochromatium vinosum soluble hydrogenase HoxU comprising SEQ ID NO: 17, or an amino acid sequence having at least 99% identity therewith; xviii) an amino acid sequence of the diaphorase moiety of Thiocapsa roseopersicina Hox1F comprising SEQ ID NO: 18, or an amino acid sequence having at least 99% identity therewith; xix) an amino acid sequence of the diaphorase moiety of Thiocapsa roseopersicina soluble hydrogenase Hox1U comprising SEQ ID NO: 19, or an amino acid sequence having at least 99% identity therewith; xx) an amino acid sequence of the diaphorase moiety of Thiocapsa roseopersicina Hox2F comprising SEQ ID NO: 20, or an amino acid sequence having at least 99% identity therewith; xxi) an amino acid sequence of the diaphorase moiety of Thiocapsa roseopersicina soluble hydrogenase Hox2U comprising SEQ ID NO: 21, or an amino acid sequence having at least 99% identity therewith; xxii) an amino acid sequence of the diaphorase moiety of Synechocystis sp. PCC 6803 HoxF comprising SEQ ID NO: 22, or an amino acid sequence having at least 99% identity therewith; xxiii) an amino acid sequence of the diaphorase moiety of Synechocystis sp. PCC6803 HoxU comprising SEQ ID NO: 23, or an amino acid sequence having at least 99% identity therewith; xxiv) an amino acid sequence of the diaphorase moiety of Synechococcus elongatus PCC 6301 HoxF comprising SEQ ID NO: 24, or an amino acid sequence having at least 99% identity therewith; xxv) an amino acid sequence of the diaphorase moiety of Synechococcus elongatus PCC 6301 HoxU comprising SEQ ID NO: 25, or an amino acid sequence having at least 99% identity therewith; xxvi) an amino acid sequence of Hydrogenophilus thermoluteolus diaphorase HoxF comprising SEQ ID NO: 26, or an amino acid sequence having at least 99% identity therewith; xxvii) an amino acid sequence of Hydrogenophilus thermoluteolus diaphorase HoxU comprising SEQ ID NO: 27, or an amino acid sequence having at least 99% identity therewith; xxviii) an amino acid sequence of Chlamydomonas reinhardtii Ferredoxin-NADP+ reductase comprising SEQ ID NO: 28, or an amino acid sequence having at least 99% identity therewith; xxix) an amino acid sequence of Anabaena Ferredoxin-NADP+ reductase comprising SEQ ID NO: 29, or an amino acid sequence having at least 99% identity therewith; xxx) an amino acid sequence of Ralstonia eutropha diaphorase HoxF comprising SEQ ID NO: 70, or an amino acid sequence having at least 99% identity therewith; and xxxi) an amino acid sequence of Ralstonia eutropha diaphorase with inactive hydrogenase comprising SEQ ID NOs: 1 and/or 2 and/or 69 and/or 31, or an amino acid sequence having at least 99% identity therewith.

8 . The method according to claim 1 , wherein the electron source comprises a second polypeptide, which is an oxidising enzyme, capable of oxidising a reductant to extract electrons, wherein the second polypeptide transfers electrons from the reductant to the fir

Assignees

• Univ Oxford Innovation Ltd

Inventors

• Vincent Kylie
• Reeve Holly
• Rowbotham Jack

Classifications

• C12P19/36 Primary

  Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate · CPC title