WO2023012819A1 - Recombinant transaminase polypeptides - Google Patents
Recombinant transaminase polypeptides Download PDFInfo
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- WO2023012819A1 WO2023012819A1 PCT/IN2022/050688 IN2022050688W WO2023012819A1 WO 2023012819 A1 WO2023012819 A1 WO 2023012819A1 IN 2022050688 W IN2022050688 W IN 2022050688W WO 2023012819 A1 WO2023012819 A1 WO 2023012819A1
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- substituted
- polar
- amino acid
- aliphatic
- alkyl
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- 102000003929 Transaminases Human genes 0.000 title claims abstract description 69
- 108090000340 Transaminases Proteins 0.000 title claims abstract description 69
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 61
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 61
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 claims abstract description 48
- 150000001412 amines Chemical class 0.000 claims abstract description 30
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 6
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 6
- 125000001931 aliphatic group Chemical group 0.000 claims description 30
- 150000001413 amino acids Chemical class 0.000 claims description 28
- -1 Aromatic amino acid Chemical class 0.000 claims description 27
- 150000001875 compounds Chemical class 0.000 claims description 26
- 125000000217 alkyl group Chemical group 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 22
- 239000000758 substrate Substances 0.000 claims description 20
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethyl sulfoxide Natural products CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 14
- 108091033319 polynucleotide Proteins 0.000 claims description 14
- 102000040430 polynucleotide Human genes 0.000 claims description 14
- 239000002157 polynucleotide Substances 0.000 claims description 14
- 125000003277 amino group Chemical group 0.000 claims description 13
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
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- MFFMDFFZMYYVKS-SECBINFHSA-N sitagliptin Chemical compound C([C@H](CC(=O)N1CC=2N(C(=NN=2)C(F)(F)F)CC1)N)C1=CC(F)=C(F)C=C1F MFFMDFFZMYYVKS-SECBINFHSA-N 0.000 claims description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 9
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 8
- 125000003118 aryl group Chemical group 0.000 claims description 8
- 125000001072 heteroaryl group Chemical group 0.000 claims description 8
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- LCDDAGSJHKEABN-MLGOLLRUSA-N Evogliptin Chemical compound C1CNC(=O)[C@@H](COC(C)(C)C)N1C(=O)C[C@H](N)CC1=CC(F)=C(F)C=C1F LCDDAGSJHKEABN-MLGOLLRUSA-N 0.000 claims description 6
- KYQCOXFCLRTKLS-UHFFFAOYSA-N Pyrazine Chemical compound C1=CN=CC=N1 KYQCOXFCLRTKLS-UHFFFAOYSA-N 0.000 claims description 6
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- 235000019000 fluorine Nutrition 0.000 claims description 6
- WIIAMRXFUJLYEF-SNVBAGLBSA-N methyl 7-[(3r)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl]-3-(trifluoromethyl)-6,8-dihydro-5h-imidazo[1,5-a]pyrazine-1-carboxylate Chemical compound C([C@@H](N)CC(=O)N1CCN2C(=NC(=C2C1)C(=O)OC)C(F)(F)F)C1=CC(F)=C(F)C=C1F WIIAMRXFUJLYEF-SNVBAGLBSA-N 0.000 claims description 6
- 229940126951 retagliptin Drugs 0.000 claims description 6
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- TUAXCHGULMWHIO-SECBINFHSA-N (3r)-3-[(2-methylpropan-2-yl)oxycarbonylamino]-4-(2,4,5-trifluorophenyl)butanoic acid Chemical group CC(C)(C)OC(=O)N[C@@H](CC(O)=O)CC1=CC(F)=C(F)C=C1F TUAXCHGULMWHIO-SECBINFHSA-N 0.000 claims description 3
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- 125000002837 carbocyclic group Chemical group 0.000 claims description 3
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- XDQLWVSUKUDAEO-UHFFFAOYSA-N methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate Chemical compound COC(=O)CC(=O)CC1=CC(F)=C(F)C=C1F XDQLWVSUKUDAEO-UHFFFAOYSA-N 0.000 claims description 2
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
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- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 claims description 2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
Definitions
- the present invention is generally related to enzyme/protein engineering. Specifically, the present invention provides recombinant transaminase polypeptides, fusion protein comprising the same, and process of designing the same. Further, the present invention provides a process of preparing chiral amines catalyzed by the recombinant transaminase polypeptides. Also, the present invention provides uses of the recombinant transaminase polypeptides.
- Transaminases are enzymes that enable transfer of an amine group from amino group donor to a keto group of an acceptor substrate.
- the cofactor, pyridoxal phosphate (PLP) facilitates catalysis with transaminase polypeptides.
- Transaminases have often been used in the synthesis of chiral amines and amino acids with high enantiomeric purity
- co- Transaminases a subclass of transaminase enzymes produce chiral aminesby resolution of the racemic amine or by asymmetric synthesis starting from the prochiral ketone, which is an amine acceptor, and uses an amine donor compound.
- co-transaminases show higher selectivity and activity towards ketoester substrates.
- Chiral amines are valuable substrates for the production of a large number of bioactive compounds with pharmacological properties.
- Various new chemical entities (NCEs) among FDA approved drugs contain chiral amine moieties.
- Sitagliptin ((3/?)-3-amino-l-[3- (trifhioromethyl)-6,8-dihydro-5H-[l,2,4]triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5- trifluorophenyl)butan-l-one) is one such oral anti-hyperglycemic drug with chiral amine moiety, and belongs to the gliptin class of anti-diabetics, characterized by their dipeptidyl- peptidase-4 (DPP -4) inhibiting activity.
- DPP -4 dipeptidyl- peptidase-4
- This enzyme-inhibiting drug is used either alone or in combination with other oral anti- hyperglycemic agents (such as metformin or a thiazolidinedione) for treatment of diabetes mellitus type 2.
- oral anti- hyperglycemic agents such as metformin or a thiazolidinedione
- Patent numbers, US219372P and US2016/0304843A1 disclose transaminases and reactions catalyzed by them. However, these documents fail to disclose S Transaminase which results in R form of chiral amines. US219372P discloses conversion using R Transaminase which results in R product. As regards Patent number US2016/0304843A1, it has been shown by the present inventors that when polypeptide disclosed, is employed on the substrate of the present invention then it resulted in an S product, which is an inactive form, and is completely different from the present invention i.e. chiral amines in R form.
- the current invention is directed to recombinant transaminases and biocatalytic synthetic routes for enantiopure chiral amines which are key intermediates for the synthesis of Sitagliptin, employing a transaminase enzyme.
- the recombinant transaminase of the invention leads to reduced cost of synthesis of enantiopure chiral amines along with high yield, purity and enantiomeric excess.
- the recombinant transaminases enable higher conversion of substrate into product requiring less enzyme loading.
- the synthesized intermediate can also be leveraged for synthesis of two other gliptins namely, Evogliptin and Retagliptin, for t h e treatment of type 2 diabetes.
- the present invention recombinant transaminase polypeptide which comprises an amino acid substitution selected from the group consisting of: i. Phe55 substituted with Cys or Met or any Aliphatic, Polar or non-polar or Aromatic amino acid; ii. Glu93 substituted with Pro or any Aliphatic, Acidic, or Polar or non-polar amino acid; iii. Glyl51 substituted with Ala, or any Aliphatic, non-polar, or Polar amino acid; iv. Thrl59 substituted with Ala, or any aliphatic or non-polar or polar amino acid; v. Glul66 substituted with Ala, or any Aliphatic, Acidic, Polar or non-polar, amino acid; vi.
- Val262 substituted with Met, or any Aliphatic, non-polar amino acid; vii. Ser298 substituted with Gly, or any Aliphatic or polar amino acid; viii. Ile427 substituted with Thr, or any Polar, or Aliphatic amino acid; ix. Val385 substituted with Leu, or He, or any non-polar or Aliphatic amino acid; x. Leu428 substituted with He, or any aliphatic or non-polar residue; xi. Leu58 substituted with Ser or Thr or any aliphatic polar amino acid; xii. Gly 154 substituted with Vai or Ala or He or Thr or any aliphatic amino acid; and xiii. Gly323 substituted with Vai or Ala or He or any aliphatic non-polar amino acid.
- the present invention provides a fusion protein comprising the same. Also, a process of designing polypeptide of the present invention is disclosed which process is performed in silico using QZyme WorkbenchTM. Furthermore, the present invention provides a process of preparing chiral amines catalyzed by the recombinant transaminase polypeptides wherein conversion rate of the recombinant transaminase polypeptides is high compared to wild type. Also, the present invention provides uses of the recombinant transaminase polypeptides.
- the present invention represents the enzymatic step required to convert beta keto ester to chiral amine DETAILED DESCRIPTION:
- amine donor refers to amine compounds such as isopropyl amine, amino acids like alanine.
- the “amine donor” can be accepted by a transaminase polypeptide, and which can supply an amino group.
- Sitagliptin refers to a compound with CAS no. 486460-32- 6. It also includes salts thereof like Sitagliptin phosphate monohydrate (CAS no. 654671-77- 9). Chemically it is (R)-3-amino-l-(3-(trifluoromethyl)-5,6-dihydro-[l,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-l-one.
- “Native” or “Naturally occurring” or “wild type” polypeptide or polynucleotide sequence refers to naturally occurring or wild-type polypeptide or polynucleotide sequence that exists in nature in an organism that can be isolated from a source in nature, and which has not been intentionally modified by human manipulation.
- Recombinant refers to polypeptide sequences or polynucleotide sequences encoding them that have been modified in their sequence to the naturally occurring or wild type sequences. These also include polypeptide sequences or polynucleotide sequences encoding them that have the same sequences as naturally occurring sequences but are obtained by means of recombinant techniques.
- enantiomeric excess refers to a parameter for measuringpurity of chiral compounds and indicates abundance of one enantiomer over theother.
- enzyme activity is defined in units. 1 unit of enzyme (U, used interchangeably herein with IU or international units) is the amount of enzyme that catalyzes the reaction of 1 pmol of substrate per minute.
- the fusion protein of the present invention comprises of tags. Tags are attached to proteins for various purposes, e.g. in order to ease purification, to assist in the proper folding in proteins, to prevent precipitation of the protein, to alter chromatographic properties, to modify the protein or to mark or label the protein.
- the tag used in the present invention is 6X His-tag.
- the substrates used for activity assay can be any substrates known for the given enzymes. Moreover, the enzymes used can be from any of the known sources.
- Percentage of sequence identity As used herein, the terms “Percentage of sequence identity,” “percent identity,” and “percent identical” are used interchangeably and herein refer to identity between a pair of polynucleotide or polypeptide sequences which is reflected as a quantitative measure. It measures the number of identical residues (“matches”) in relation to the length of the alignment of the polynucleotide or polypeptide sequences compared. "Percent identity” as used herein can be measured by an indigenous technology QZyme WorkbenchTM . This technology is a fully automated proprietary in silico protein engineering platform, evolved by integrating open- source computational chemistry and biology/bioinformatics tools in combination with customized algorithms and scripts.
- this software is capable of tackling several important aspects of protein modelling and engineering including, but not limited to, structural refinement, ligand docking, conformational sampling, estimating substrate binding affinity, modelling catalytic reaction, identifying mutable hotspots, further hotspot optimization.
- Transaminase and “Aminotransferase” are used interchangeably herein and refer to enzymes that catalyze the transfer of an amino group from an amino donor to an amino acceptor.
- This class of enzymes belongs to pyridoxalphosphate dependent aminotransferase family. It includes co-transaminases, a class of enzymes that belong to Class III aminotransferases which catalyze the transfer of an amino group from a non-a position amino acid, or an amine compound with no carboxylic group, to an amino acceptor, co-transaminases have been often used in synthesis of various building blocks in chemical and pharmaceutical industry.
- the present invention encompasses recombinant transaminase polypeptides and a process of preparing chiral amines catalyzed by the recombinant transaminase polypeptides.
- Transaminase polypeptides catalyze the conversion of Beta-ketoester compounds to enantiopure chiral amines.
- co-transaminases show higher selectivity and activity towards ketoester substrates.
- the recombinant transaminase polypeptides disclosed in the current invention enable highly selective, improved catalytic synthesis of chiral amines with high enantiomeric purity, high product yield and at high conversion rate. Further, the conversion of the substrate of present invention to R-product is not well characterized in prior art using the recombinant transaminase polypeptides of the present invention.
- the recombinant transaminase polypeptides disclosed herein have high enzymatic activity compared to the naturally occurring or a base variant.
- Transaminases found in nature lack the substrate specificity and selectivity to enable catalysis with high product yield and purity.
- Chiral amines in general, are compounds that are of immense interest and value across diverse fields including agriculture, pharmaceutical, nutraceutical domains.
- Transaminase polypeptides of the invention enable efficient synthesis of chiral amines.
- One such chiral amine BOC butanoic acid ((R)-3-(tert- Butoxycarbonylamino)-4- (2,4,5-trifluorophenyl)butanoic acid.)
- BOC butanoic acid ((R)-3-(tert- Butoxycarbonylamino)-4- (2,4,5-trifluorophenyl)butanoic acid.)
- gliptins like Sitagliptin, Evogliptin, and Retagliptin.
- the present invention provides recombinant transaminase polypeptide which comprises an amino acid substitution selected from the group consisting of: i. Phe55 substituted with Cys or Met or any Aliphatic, Polar or non-polar or Aromatic amino acid; ii.
- Glu93 substituted with Pro or any Aliphatic, Acidic, or Polar or non-polar amino acid iii. Glyl51 substituted with Ala, or any Aliphatic, non-polar, or Polar amino acid; iv. Thrl59 substituted with Ala, or any aliphatic or non-polar or polar amino acid; v. Glul66 substituted with Ala, or any Aliphatic, Acidic, Polar or non-polar, amino acid; vi. Val262 substituted with Met, or any Aliphatic, non-polar amino acid; vii. Ser298 substituted with Gly, or any Aliphatic or polar amino acid; viii. Ile427 substituted with Thr, or any Polar, or Aliphatic amino acid; ix.
- Val385 substituted with Leu, or He, or any non-polar or Aliphatic amino acid
- x. Leu428 substituted with He, or any aliphatic or non-polar residue
- xi. Leu58 substituted with Ser or Thr or any aliphatic polar amino acid
- xii. Gly 154 substituted with Vai or Ala or He or Thr or any aliphatic amino acid
- xiii. Gly323 substituted with Vai or Ala or He or any aliphatic non-polar amino acid.
- said polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 to 45.
- said polypeptide is represented amino acid sequences selected from SEQ ID No. 1 to 45.
- said polypeptide further comprises an amino acid substitution selected from the group consisting of: Phe55Cys, Phe55Met, Glu93Pro, Glyl51Ala, Thrl59Ala, Glul66Ala, Val262Met, Ser298Gly, Ile427Thr, Val385Leu, Val385Ile and Leu428Ile, Leu58Ser, Leu58Thr, Glyl54Val, Glyl54Ala, Glyl54Ile, Gly323Val, GLy323Ala, GLy323Ile.
- the present invention provides a polynucleotide encoding the recombinant transaminase polypeptide of the present invention, wherein said polynucleotide is selected from SEQ ID Nos. 46-56.
- the present invention provides a fusion protein comprising the recombinant transaminase polypeptide of the present invention and 6X His-tag.
- the present invention provides an expression vector comprising the polynucleotide of the present invention.
- the present invention provides a host comprising the expression vector of the present invention, wherein said host cell in a bacterial cell.
- the present invention provides a process of preparing a compound of structural formula I,
- R1 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-Ci-2 alkyl, carbocyclic, heterocyclic, optionally being unsubstituted or substituted with one to five fluorine, or any halogen; and
- R2 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-Ci-2 alkyl, optionally being unsubstituted or substituted; wherein the process comprising the step of contacting a prochiral ketone compound of structural Formula (II):
- X is OR2;
- R1 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-Ci-2 alkyl, carbocylclic, heterocyclic, optionally being unsubstituted or substituted with one to five fluorine, or any halogen; and
- R2 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-Ci-2 alkyl, optionally being unsubstituted or substituted; with the transaminase polypeptide of the present invention; in the presence of an amino group donor; and wherein the conversion is such that the product obtained has enantiomeric form “R”.
- R1 in the compound of structural formula I is a benzyl group, and wherein the phenyl group of benzyl is unsubstituted or substituted withone to five fluorines.
- the compound of formula I is (R)-3-amino-4- (2,4,5- trifluorophenyl) butyric acid methyl ester.
- the compound of formula I is produced in the range of 60- 100% enantiomeric excess.
- the amount of recombinant transaminases required for conversion of compound of structural formula II to compound of formula I is 1.5 mg/mL
- the rate of conversion of compound of formula II to compound of formula I ranges from 10% to 95%.
- the amino group donor is selected from o- Xylylenediamine and isopropyl amine.
- said process further comprises a step of protecting the amino group by an amino protecting group selected from formyl, acetyl, trifluoro acetyl, benzyl, benzyloxy carbonyl ("CBZ”), tert-butoxy carbonyl (“BOC”), trimethylsilyl (“TMS”), 2-trimethylsilyl- ethanesulfonyl (“SES”), trityl and substituted trityl groups, allyloxycarbonyl, 9- fluorenylmethyloxy carbonyl (“FMOC”), and nitroveratryloxycarbonyl (“NVOC”).
- an amino protecting group selected from formyl, acetyl, trifluoro acetyl, benzyl, benzyloxy carbonyl (“CBZ”), tert-butoxy carbonyl (“BOC”), trimethylsilyl (“TMS”), 2-trimethylsilyl- ethanesulfonyl (“SES”), tr
- the amino protecting group is a BOC protecting group.
- the BOC protected chiral amine is (R)-3-(tert- Butoxycarbonylamino)-4-(2,4,5-trifluorophenyl)butanoic acid.
- the organic solvent is selected from dimethylsulfoxide (DMSO), Dimethylformamide (DMF), methyl tert- butyl ether (MTBE), isopropyl acetate, methanol, ethanol or propanol.
- the solvent is a mixture of water and dimethylsulfoxide (DMSO),
- the prochiral ketone of structural formula II is 3-oxo-4-(2,4,5 trifluorophenyl)butyricacid methyl ester.
- the process of preparing the compound of formula I further comprises the step of reacting the compound of formula I with 3-(trifluoromethyl)-5, 6,7,8- tetrahydro [l,2,4]triazolo[4,3-a]pyrazine, 3-(trifluoromethyl)-6,8-dihydro-5/Z-imidazo[ 1 ,5- a]pyrazine, and3-[(2-methylpropan-2-yl)oxymethyl]piperazin-2-one to make Sitagliptin, Retagliptin, and Evogliptin, respectively.
- the present invention provides a method of designing the recombinant transaminase polypeptide of the present invention, wherein said method is performed in silico using QZyme WorkbenchTM and comprising the steps of
- the present invention provides use of the recombinant transaminase polypeptide in manufacturing of chiral amines, wherein said chiral amines are essential intermediates in production of pharmaceutical drugs selected from Sitagliptin, Evogliptin and Retagliptin, which treats diabetes mellitus type 2.
- recombinant transaminase polypeptide in the form of whole cell, crude extract, isolated polypeptide, or purified polypeptide alone or in combination with another recombinant transaminase polypeptide.
- the present invention provides a method of treating diabetes mellitus type 2, wherein said method comprising administering a subject a drug manufactured using intermediates obtained from the process as defined in the present invention.
- An in silico enzyme engineering framework was used (QZyme WorkbenchTM) to engineer the transaminase enzymes to achieve high catalytic efficiency.
- the framework carries out different aspects of in silico protein engineering including structural refinement and modelling, ligand docking, conformational sampling, estimating substrate binding affinity, modelling catalytic reaction, identifying mutable hotspots, further hotspot optimization.
- modelled structure of the protein was modelled to an appropriate oligomeric state. Additional structural refinement was carried out in this second step to ensure that the modelled structure satisfied catalytically competent conformation (open vs closed state).
- the modelled structure thus obtained was usedto model the near- attack conformation of the substrate in the enzyme active site (Michaelis complex) by implementing several docking algorithms. In this step, the bottle neck of the enzymatic conversion of the substrate was also determined.
- the next step included discovery of key functional residues and evaluation of their mutability in an effort to improve the enzyme function.
- the step further incorporated a rapid screening method to know the contribution of each amino acid and all possible amino acid substitutions to the enzyme’s function and stability.
- Sequence analysis as well as contact score analysis was given priority for selection of hotspots. Hotspots were selected based on partial conserved residues obtained through Delta- BLAST using NR database. The conserved residues obtained through contact score analysis was selected. All the conserved residues were neglected whereas the partially conserved residues were checked to create a focused library.
- stability analysis was carried out for selecting hotspots. The common mutations obtained through sequence alignment, and stability analysis were shortlisted for the designing step. Thus, a huge library of variants is created, followedby creation of a focused library.
- the rate-limiting step (previously referred to as the bottleneck) was modelled for each of the variants belonging to the focused library, and compared with the variants of the wild type enzyme. In addition to that, binding energy calculations were then performed for all the variants from the focused library.
- the purpose of the designing step is to reduce the false positives and to increase the quality of the focused library.
- top variants were shortlisted for further validation through lab experiments.
- the mutations in the present invention are designed from the wild type sequence of UniProt ID: Q1GD43.
- Table 2 below, lists the polynucleotides of the present invention and the recombinant transaminase polypeptides which they encode: Table 2:
- the plasmids of wildtype and mutants were transformed into E. coli BL21(DE3) competent cell. Single colony was inoculated to LB broth containing ampicillin (100 pg/mL). 2% of the inoculum was inoculated to TB broth containing ampicillin (100 pg/mL). Cells were grown at 37°C till ODeoo reaches 0.8- 1.0. cultures were induced with 0.5mM IPTG and grown further overnight at 25°C. Expressions were analysed via SDS-PAGE and total protein was estimated by Bradford method with BSA (Bovine Serum Albumin) as standard.
- BSA Bovine Serum Albumin
- Pellet was resuspended in 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl and 0.1 mM PLP and lysed by sonication. Lysate was centrifuged and soluble fraction recovered was incubated with Ni-NTA beads pre-equilibrated with Tris buffer (pH 7.5) at 4 °C. Ni-NTA beads were subsequently washed with 50 mM Tris buffer (pH 7.5) containing 300 mM NaCl, 5 mM imidazole and 0.1 mM PLP.
- the assay is performed with a total reaction mixture of 1.0 ml in a 2.0 ml vial.
- the reaction is carried out with an optimized Substrate: Amine donor ratio of 1 :5.
- the working concentration of the reaction components is given below:
- Reaction mixture was incubated at 40 °C for 48 h with continuous mixing at 1000 rpm in a thermomixer
- Lyophilized samples were treated with 500 pL AcCl at 40 °C for 2 hours. The AcCl was subsequently left to evaporate at 50 °C for approx. 30 minutes. The mostly solidified (gummy viscous) residue was extracted with 50 pL MeCN which was done by repeated washing of the solid material. The usually orange suspension was transferred into micro-Eppendorf tubes and centrifuged. The supernatant was measured using the chiral GC.
- GC analysis was performed on a BGB175 column gamma cylcodextrixn column (50% 2,3- diacetyl-6-tert-butyldimethylsilyl-gamma-cyclodextrin dissolved in BGB-1701 [14% cyanopropylphenyl-, 86% methylpolysiloxane]).
- the gas flow was set to Iml/min, detection was done using an FID detector.
- the table 6 below includes results for recombinant transaminase polypeptides which were tested in-silico to determine its efficiency in converting a product in its R and S forms. The test conducted and results obtained extends to all the 45 polypeptides described in Table 1.
- Table: 6 shows in silico conversion efficiency of the transaminases of the present invention into R and S form of product:
- the table 7 below includes results for recombinant transaminase polypeptides which were tested in vitro to determine its efficiency in converting a product in its R and S forms and also represents results for product conversion rate. The test conducted and results obtained extends to all the 45 polypeptides described in Table 1.
- Table 7 shows in vitro efficiency of the transaminases of the present invention.
Abstract
The present invention provides recombinant transaminase polypeptides, fusion protein comprising the same and process of designing the same. Further, the present invention provides a process of preparing chiral amines catalyzed by the recombinant transaminase polypeptides. Also, the present invention provides uses of the recombinant transaminase polypeptides. The recombinant transaminases of the invention lead to reduced cost of synthesis of enantiopure chiral amines along with high yield and purity.
Description
Recombinant Transaminase Polypeptides
TECHNICAL FIELD:
The present invention is generally related to enzyme/protein engineering. Specifically, the present invention provides recombinant transaminase polypeptides, fusion protein comprising the same, and process of designing the same. Further, the present invention provides a process of preparing chiral amines catalyzed by the recombinant transaminase polypeptides. Also, the present invention provides uses of the recombinant transaminase polypeptides.
BACKGROUND ART:
Transaminases are enzymes that enable transfer of an amine group from amino group donor to a keto group of an acceptor substrate. The cofactor, pyridoxal phosphate (PLP) facilitates catalysis with transaminase polypeptides. Transaminases have often been used in the synthesis of chiral amines and amino acids with high enantiomeric purity, co- Transaminases, a subclass of transaminase enzymes produce chiral aminesby resolution of the racemic amine or by asymmetric synthesis starting from the prochiral ketone, which is an amine acceptor, and uses an amine donor compound. Amongst different classes of transaminases, co-transaminases show higher selectivity and activity towards ketoester substrates.
Chiral amines are valuable substrates for the production of a large number of bioactive compounds with pharmacological properties. Various new chemical entities (NCEs) among FDA approved drugs contain chiral amine moieties. Sitagliptin ((3/?)-3-amino-l-[3- (trifhioromethyl)-6,8-dihydro-5H-[l,2,4]triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5- trifluorophenyl)butan-l-one) is one such oral anti-hyperglycemic drug with chiral amine moiety, and belongs to the gliptin class of anti-diabetics, characterized by their dipeptidyl- peptidase-4 (DPP -4) inhibiting activity. It was developed and marketed by Merck & Co. This enzyme-inhibiting drug is used either alone or in combination with other oral anti- hyperglycemic agents (such as metformin or a thiazolidinedione) for treatment of diabetes mellitus type 2.
The original synthesis of Sitagliptin required the asymmetric hydrogenation of an enamine at high pressure using a rhodium-based chiral catalyst. The major drawbacks in this process are inadequate stereoselectivity and the rhodium catalyst contamination in the downstream processing, necessitating additional purification steps, at the expense of both enantiomeric excess (“ee”) and chemical purity.
'There are currently existing technologies leveraging a transaminase scaffold along with various protein engineering techniques, which have improved the efficiency of Sitagliptin manufacturing by enzymatic catalysis.
The prior approaches to synthesize chiral amines use expensive metal catalysts along with extreme reaction conditions such as high pressure and high temperature, leading to lower product yields with longer reaction times. Moreover, these processes entail cumbersome purification along with generation of higher waste components rendering these processes non-environment friendly.
Patent numbers, US219372P and US2016/0304843A1, disclose transaminases and reactions catalyzed by them. However, these documents fail to disclose S Transaminase which results in R form of chiral amines. US219372P discloses conversion using R Transaminase which results in R product. As regards Patent number US2016/0304843A1, it has been shown by the present inventors that when polypeptide disclosed, is employed on the substrate of the present invention then it resulted in an S product, which is an inactive form, and is completely different from the present invention i.e. chiral amines in R form.
Use of substrate specific, selective transaminases is useful in industrial scale production of several compounds including compounds of pharmaceutical, nutraceutical interest. The current invention is directed to recombinant transaminases and biocatalytic synthetic routes for enantiopure chiral amines which are key intermediates for the synthesis of Sitagliptin, employing a transaminase enzyme. The recombinant transaminase of the invention leads to reduced cost of synthesis of enantiopure chiral amines along with high yield, purity and enantiomeric excess. The recombinant transaminases enable higher conversion of substrate into product requiring less enzyme loading. The synthesized intermediate can also be leveraged for synthesis of two other gliptins namely, Evogliptin and Retagliptin, for t h e treatment of type 2 diabetes.
SUMMARY:
The present invention recombinant transaminase polypeptide which comprises an amino acid substitution selected from the group consisting of: i. Phe55 substituted with Cys or Met or any Aliphatic, Polar or non-polar or Aromatic amino acid; ii. Glu93 substituted with Pro or any Aliphatic, Acidic, or Polar or non-polar amino acid; iii. Glyl51 substituted with Ala, or any Aliphatic, non-polar, or Polar amino acid; iv. Thrl59 substituted with Ala, or any aliphatic or non-polar or polar amino acid; v. Glul66 substituted with Ala, or any Aliphatic, Acidic, Polar or non-polar, amino acid;
vi. Val262 substituted with Met, or any Aliphatic, non-polar amino acid; vii. Ser298 substituted with Gly, or any Aliphatic or polar amino acid; viii. Ile427 substituted with Thr, or any Polar, or Aliphatic amino acid; ix. Val385 substituted with Leu, or He, or any non-polar or Aliphatic amino acid; x. Leu428 substituted with He, or any aliphatic or non-polar residue; xi. Leu58 substituted with Ser or Thr or any aliphatic polar amino acid; xii. Gly 154 substituted with Vai or Ala or He or Thr or any aliphatic amino acid; and xiii. Gly323 substituted with Vai or Ala or He or any aliphatic non-polar amino acid.
Further, the present invention provides a fusion protein comprising the same. Also, a process of designing polypeptide of the present invention is disclosed which process is performed in silico using QZyme Workbench™. Furthermore, the present invention provides a process of preparing chiral amines catalyzed by the recombinant transaminase polypeptides wherein conversion rate of the recombinant transaminase polypeptides is high compared to wild type. Also, the present invention provides uses of the recombinant transaminase polypeptides.
BRIEF DESCRIPTION OF FIGURES AND DRAWINGS:
The accompanying drawings illustrate some of the embodiments of the present invention and, together with the descriptions, serve to explain the invention. The drawing (s) has been provided by way of illustration and not by way of limitation. Figure 1 Shows the scheme of synthesis of BOC Butanoic acid ((R)-3-(tert-
Butoxycarbonylamino)-4-(2,4,5-trifluorophenyl)butanoic acid). The present invention represents the enzymatic step required to convert beta keto ester to chiral amine DETAILED DESCRIPTION:
The present invention is now described with reference to the tables/figures and specific embodiments, including the best mode contemplated by the inventors for carrying out the invention. This description is not meant to be construed in a limiting sense, as various alternate embodiments of the invention will become apparent to persons skilled in the art, upon reference to the description of the invention. It is therefore contemplated that such alternative embodiments form part of the present invention. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or
polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Some of the terms are defined briefly here below; the definitions should not be construed in a limiting sense.
The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.
The term “plurality” as used herein is defined as “one, or more than one”. Accordingly, the terms “one”, “at least one” would all fall under the definition of “plurality”.
Whether or not a certain feature or element was limited to being used only once, either way it may still be referred to as “one or more features” or “one or more elements” or “at least one feature” or “at least one element”.
Definitions:
As used herein the term "amine donor" refers to amine compounds such as isopropyl amine, amino acids like alanine. The “amine donor” can be accepted by a transaminase polypeptide, and which can supply an amino group.
As used herein the term "Sitagliptin" refers to a compound with CAS no. 486460-32- 6. It also includes salts thereof like Sitagliptin phosphate monohydrate (CAS no. 654671-77- 9). Chemically it is (R)-3-amino-l-(3-(trifluoromethyl)-5,6-dihydro-[l,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-l-one.
As used herein the term "Native" or "Naturally occurring" or "wild type" polypeptide or polynucleotide sequence refers to naturally occurring or wild-type polypeptide or polynucleotide sequence that exists in nature in an organism that can be isolated from a source in nature, and which has not been intentionally modified by human manipulation.
As used herein the term "Recombinant" refers to polypeptide sequences or polynucleotide sequences encoding them that have been modified in their sequence to the naturally occurring or wild type sequences. These also include polypeptide sequences or polynucleotide sequences encoding them that have the same sequences as naturally occurring sequences but are obtained by means of recombinant techniques. As used herein the term "enantiomeric excess" refers to a parameter for measuringpurity of chiral compounds and indicates abundance of one enantiomer over theother.
As used herein, "enzyme activity" is defined in units. 1 unit of enzyme (U, used interchangeably herein with IU or international units) is the amount of enzyme that catalyzes the reaction of 1 pmol of substrate per minute.
The fusion protein of the present invention comprises of tags. Tags are attached to proteins for various purposes, e.g. in order to ease purification, to assist in the proper folding in proteins, to prevent precipitation of the protein, to alter chromatographic properties, to modify the protein or to mark or label the protein. The tag used in the present invention is 6X His-tag.
The substrates used for activity assay can be any substrates known for the given enzymes. Moreover, the enzymes used can be from any of the known sources.
As used herein, the terms “Percentage of sequence identity,” “percent identity,” and “percent identical” are used interchangeably and herein refer to identity between a pair of polynucleotide or polypeptide sequences which is reflected as a quantitative measure. It measures the number of identical residues (“matches”) in relation to the length of the alignment of the polynucleotide or polypeptide sequences compared. "Percent identity" as used herein can be measured by an indigenous technology QZyme Workbench™ . This technology is a fully automated proprietary in silico protein engineering platform, evolved by integrating open- source computational chemistry and biology/bioinformatics tools in combination with customized algorithms and scripts. Further, this software is capable of tackling several important aspects of protein modelling and engineering including, but not limited to, structural refinement, ligand docking, conformational sampling, estimating substrate binding affinity, modelling catalytic reaction, identifying mutable hotspots, further hotspot optimization.
As used herein, the terms "Transaminase" and "Aminotransferase" are used interchangeably herein and refer to enzymes that catalyze the transfer of an amino group from an amino donor to an amino acceptor. This class of enzymes belongs to pyridoxalphosphate dependent aminotransferase family. It includes co-transaminases, a class of enzymes that belong to Class III aminotransferases which catalyze the transfer of an amino group from a non-a position amino acid, or an amine compound with no carboxylic group, to an amino acceptor, co-transaminases have been often used in synthesis of various building blocks in chemical and pharmaceutical industry.
The present invention encompasses recombinant transaminase polypeptides and a process of preparing chiral amines catalyzed by the recombinant transaminase polypeptides.
Transaminase polypeptides catalyze the conversion of Beta-ketoester compounds to enantiopure chiral amines. Among different classes of transaminases, co-transaminases show higher selectivity and activity towards ketoester substrates.
The recombinant transaminase polypeptides disclosed in the current invention enable
highly selective, improved catalytic synthesis of chiral amines with high enantiomeric purity, high product yield and at high conversion rate. Further, the conversion of the substrate of present invention to R-product is not well characterized in prior art using the recombinant transaminase polypeptides of the present invention. The recombinant transaminase polypeptides disclosed herein have high enzymatic activity compared to the naturally occurring or a base variant.
Transaminases found in nature lack the substrate specificity and selectivity to enable catalysis with high product yield and purity.
Chiral amines, in general, are compounds that are of immense interest and value across diverse fields including agriculture, pharmaceutical, nutraceutical domains.
Transaminase polypeptides of the invention enable efficient synthesis of chiral amines. One such chiral amine, BOC butanoic acid ((R)-3-(tert- Butoxycarbonylamino)-4- (2,4,5-trifluorophenyl)butanoic acid.), is an intermediate in the synthesis of gliptins like Sitagliptin, Evogliptin, and Retagliptin. In a first aspect, the present invention provides recombinant transaminase polypeptide which comprises an amino acid substitution selected from the group consisting of: i. Phe55 substituted with Cys or Met or any Aliphatic, Polar or non-polar or Aromatic amino acid; ii. Glu93 substituted with Pro or any Aliphatic, Acidic, or Polar or non-polar amino acid; iii. Glyl51 substituted with Ala, or any Aliphatic, non-polar, or Polar amino acid; iv. Thrl59 substituted with Ala, or any aliphatic or non-polar or polar amino acid; v. Glul66 substituted with Ala, or any Aliphatic, Acidic, Polar or non-polar, amino acid; vi. Val262 substituted with Met, or any Aliphatic, non-polar amino acid; vii. Ser298 substituted with Gly, or any Aliphatic or polar amino acid; viii. Ile427 substituted with Thr, or any Polar, or Aliphatic amino acid; ix. Val385 substituted with Leu, or He, or any non-polar or Aliphatic amino acid; x. Leu428 substituted with He, or any aliphatic or non-polar residue; xi. Leu58 substituted with Ser or Thr or any aliphatic polar amino acid; xii. Gly 154 substituted with Vai or Ala or He or Thr or any aliphatic amino acid; andxiii. Gly323 substituted with Vai or Ala or He or any aliphatic non-polar amino acid.
In one embodiment, said polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 to 45.
In another embodiment, said polypeptide is represented amino acid sequences selected from SEQ ID No. 1 to 45.
In yet another embodiment, said polypeptide further comprises an amino acid substitution selected from the group consisting of: Phe55Cys, Phe55Met, Glu93Pro, Glyl51Ala, Thrl59Ala, Glul66Ala, Val262Met, Ser298Gly, Ile427Thr, Val385Leu, Val385Ile and Leu428Ile, Leu58Ser, Leu58Thr, Glyl54Val, Glyl54Ala, Glyl54Ile, Gly323Val, GLy323Ala, GLy323Ile.
In a second aspect, the present invention provides a polynucleotide encoding the recombinant transaminase polypeptide of the present invention, wherein said polynucleotide is selected from SEQ ID Nos. 46-56.
In a third aspect, the present invention provides a fusion protein comprising the recombinant transaminase polypeptide of the present invention and 6X His-tag.
In a fourth aspect, the present invention provides an expression vector comprising the polynucleotide of the present invention.
In a fifth aspect, the present invention provides a host comprising the expression vector of the present invention, wherein said host cell in a bacterial cell.
In a sixth aspect, the present invention provides a process of preparing a compound of structural formula I,
(I) having the indicated stereochemical configuration at the stereogenic center markedwith an *; in an enantiomeric excess of at least 70 % over the opposite enantiomer, wherein X is OR2;
R1 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-Ci-2 alkyl, carbocyclic, heterocyclic, optionally being unsubstituted or substituted with one to five fluorine, or any halogen; and
R2 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-Ci-2 alkyl, optionally being unsubstituted or substituted; wherein the process comprising the step of contacting a prochiral ketone compound of structural Formula (II):
(II)
X is OR2;
R1 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-Ci-2 alkyl, carbocylclic, heterocyclic, optionally being unsubstituted or substituted with one to five fluorine, or any halogen; and
R2 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-Ci-2 alkyl, optionally being unsubstituted or substituted; with the transaminase polypeptide of the present invention; in the presence of an amino group donor; and wherein the conversion is such that the product obtained has enantiomeric form “R”.
In an embodiment, R1 in the compound of structural formula I is a benzyl group, and wherein the phenyl group of benzyl is unsubstituted or substituted withone to five fluorines.
In another embodiment, the compound of formula I is (R)-3-amino-4- (2,4,5- trifluorophenyl) butyric acid methyl ester.
In yet another embodiment, the compound of formula I is produced in the range of 60- 100% enantiomeric excess.
In an embodiment, the amount of recombinant transaminases required for conversion of compound of structural formula II to compound of formula I is 1.5 mg/mL
In another embodiment, the rate of conversion of compound of formula II to compound of formula I ranges from 10% to 95%.
In yet another embodiment, the amino group donor is selected from o- Xylylenediamine and isopropyl amine.
In an embodiment, said process further comprises a step of protecting the amino group by an amino protecting group selected from formyl, acetyl, trifluoro acetyl, benzyl, benzyloxy carbonyl ("CBZ"), tert-butoxy carbonyl ("BOC"), trimethylsilyl ("TMS"), 2-trimethylsilyl- ethanesulfonyl (“SES”), trityl and substituted trityl groups, allyloxycarbonyl, 9- fluorenylmethyloxy carbonyl ("FMOC"), and nitroveratryloxycarbonyl (“NVOC”).
In another embodiment, the amino protecting group is a BOC protecting group.
In yet another embodiment, the BOC protected chiral amine is (R)-3-(tert- Butoxycarbonylamino)-4-(2,4,5-trifluorophenyl)butanoic acid.
In an embodiment, the organic solvent is selected from dimethylsulfoxide (DMSO), Dimethylformamide (DMF), methyl tert- butyl ether (MTBE), isopropyl acetate, methanol, ethanol or propanol.
In another embodiment, the solvent is a mixture of water and dimethylsulfoxide (DMSO),
In another embodiment, the prochiral ketone of structural formula II is 3-oxo-4-(2,4,5 trifluorophenyl)butyricacid methyl ester.
In an embodiment, the process of preparing the compound of formula I further comprises the step of reacting the compound of formula I with 3-(trifluoromethyl)-5, 6,7,8- tetrahydro [l,2,4]triazolo[4,3-a]pyrazine, 3-(trifluoromethyl)-6,8-dihydro-5/Z-imidazo[ 1 ,5- a]pyrazine, and3-[(2-methylpropan-2-yl)oxymethyl]piperazin-2-one to make Sitagliptin, Retagliptin, and Evogliptin, respectively.
In a seventh aspect, the present invention provides a method of designing the recombinant transaminase polypeptide of the present invention, wherein said method is performed in silico using QZyme Workbench™ and comprising the steps of
- structural refinement and modelling;
- ligand docking;
- conformational sampling;
- estimating substrate binding affinity;
- modelling catalytic reaction;
- identifying mutable hotspots and further optimizing the hotspot, and wherein said method provides an efficient recombinant transaminase polypeptide of the present invention having increased catalytic activity.
In eighth aspect, the present invention provides use of the recombinant transaminase polypeptide in manufacturing of chiral amines, wherein said chiral amines are essential intermediates in production of pharmaceutical drugs selected from Sitagliptin, Evogliptin and Retagliptin, which treats diabetes mellitus type 2.
In an embodiment, recombinant transaminase polypeptide in the form of whole cell, crude extract, isolated polypeptide, or purified polypeptide alone or in combination with another recombinant transaminase polypeptide.
In ninth aspect, the present invention provides a method of treating diabetes mellitus type 2, wherein said method comprising administering a subject a drug manufactured using intermediates obtained from the process as defined in the present invention.
The present disclosure with reference to the following accompanying examples describes the present invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. It is understood that the examples are provided for the purpose of illustrating the invention only, and are not intended to limit the scope of the invention in any way.
EXAMPLES:
Example 1: Method of Incorporating Mutations
An in silico enzyme engineering framework was used (QZyme Workbench™) to engineer the transaminase enzymes to achieve high catalytic efficiency. The framework carries out different aspects of in silico protein engineering including structural refinement and modelling, ligand docking, conformational sampling, estimating substrate binding affinity, modelling catalytic reaction, identifying mutable hotspots, further hotspot optimization.
Three-dimensional structure of the protein (co-transaminase) was modelled to an appropriate oligomeric state. Additional structural refinement was carried out in this second step to ensure that the modelled structure satisfied catalytically competent conformation (open vs closed state). The modelled structure thus obtained was usedto model the near- attack conformation of the substrate in the enzyme active site (Michaelis complex) by implementing several docking algorithms. In this step, the bottle neck of the enzymatic conversion of the substrate was also determined. In particular, three areas were investigated in detail: a) Dynamics of Michaelis complex through classical molecular dynamics simulation to assess the stability of Michaelis complex; b) Non-equilibrium molecular dynamics simulation to study the substrate entry, product exit and to estimate associated free energy barriers, and c) The rate-limiting step of the reaction using hybrid quantum mechanics/ molecular mechanics (QM/MM) approach. The energy barriers associated with each step are further compared to identify the step that has the highest activation barrier (rate-limiting).
To address the bottleneck of enzymatic conversion identified in the previous step, the next step included discovery of key functional residues and evaluation of their mutability in an effort to improve the enzyme function. The step further incorporated a rapid screening method to know the contribution of each amino acid and all possible amino acid substitutions to the enzyme’s function and stability. Sequence analysis as well as contact score analysis was given priority for selection of hotspots. Hotspots were selected
based on partial conserved residues obtained through Delta- BLAST using NR database. The conserved residues obtained through contact score analysis was selected. All the conserved residues were neglected whereas the partially conserved residues were checked to create a focused library. Apart from sequence alignment, stability analysis was carried out for selecting hotspots. The common mutations obtained through sequence alignment, and stability analysis were shortlisted for the designing step. Thus, a huge library of variants is created, followedby creation of a focused library.
In the designing step, the rate-limiting step (previously referred to as the bottleneck) was modelled for each of the variants belonging to the focused library, and compared with the variants of the wild type enzyme. In addition to that, binding energy calculations were then performed for all the variants from the focused library. The purpose of the designing step is to reduce the false positives and to increase the quality of the focused library. As a final outcome, top variants were shortlisted for further validation through lab experiments. TABLE 1: Lists of the Mutations in Recombinant Transaminase Polypeptides:
# The mutations in the present invention are designed from the wild type sequence of UniProt ID: Q1GD43.
Table 2 below, lists the polynucleotides of the present invention and the recombinant transaminase polypeptides which they encode: Table 2:
Example 2: Protein overexpression:
The plasmids of wildtype and mutants were transformed into E. coli BL21(DE3) competent cell. Single colony was inoculated to LB broth containing ampicillin (100 pg/mL). 2% of the inoculum was inoculated to TB broth containing ampicillin (100 pg/mL). Cells were grown at 37°C till ODeoo reaches 0.8- 1.0. cultures were induced with 0.5mM IPTG and grown further overnight at 25°C. Expressions were analysed via SDS-PAGE and total protein was estimated by Bradford method with BSA (Bovine Serum Albumin) as standard.
Example 3: Protein purification:
Pellet was resuspended in 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl and 0.1 mM PLP and lysed by sonication. Lysate was centrifuged and soluble fraction recovered was incubated with Ni-NTA beads pre-equilibrated with Tris buffer (pH 7.5) at 4 °C. Ni-NTA
beads were subsequently washed with 50 mM Tris buffer (pH 7.5) containing 300 mM NaCl, 5 mM imidazole and 0.1 mM PLP. Beads are again washed with 50 mM Tris buffer (pH 7.5) containing 300 mM NaCl, 10 mM imidazole and 0.1 mM PLP. Protein elution was carried out using 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl, 200 mM imidazole and 1.0 mM PLP. Elution fraction obtained was desalted via buffer exchange with 75 mM Tris buffer (pH
7.5) containing 150 mM NaCl using 30 kDa Molecular weight centrifugal concentrator.
Example 4: Biochemical Assay: a. Assay Conditions
The assay is performed with a total reaction mixture of 1.0 ml in a 2.0 ml vial. The reaction is carried out with an optimized Substrate: Amine donor ratio of 1 :5. The working concentration of the reaction components is given below:
Table 3:
Reaction mixture was incubated at 40 °C for 48 h with continuous mixing at 1000 rpm in a thermomixer
Example 4: Quantitative Analysis of assay a. RP-HPLC
An aliquot (100 pl) was quenched with 6 N HC1 (10 pl) and diluted with 220 pl methanol (containing 3.75 mM benzoic acid) and shaken. The mixture was filtered over a syringe filter (0.2 pm) and subjected to Cl 8 HPLC measurement.
Table 4:
b. CHIRAL-GC
Lyophilized samples were treated with 500 pL AcCl at 40 °C for 2 hours. The AcCl was subsequently left to evaporate at 50 °C for approx. 30 minutes. The mostly solidified (gummy viscous) residue was extracted with 50 pL MeCN which was done by repeated washing of the solid material. The usually orange suspension was transferred into micro-Eppendorf tubes and centrifuged. The supernatant was measured using the chiral GC.
Table 5: Temperature profile for Chiral GC
GC analysis was performed on a BGB175 column gamma cylcodextrixn column (50% 2,3- diacetyl-6-tert-butyldimethylsilyl-gamma-cyclodextrin dissolved in BGB-1701 [14%
cyanopropylphenyl-, 86% methylpolysiloxane]). The gas flow was set to Iml/min, detection was done using an FID detector.
Results:
The table 6 below includes results for recombinant transaminase polypeptides which were tested in-silico to determine its efficiency in converting a product in its R and S forms. The test conducted and results obtained extends to all the 45 polypeptides described in Table 1.
Table: 6 shows in silico conversion efficiency of the transaminases of the present invention into R and S form of product:
The table 7 below includes results for recombinant transaminase polypeptides which were tested in vitro to determine its efficiency in converting a product in its R and S forms and also represents results for product conversion rate. The test conducted and results obtained extends to all the 45 polypeptides described in Table 1.
Table 7 shows in vitro efficiency of the transaminases of the present invention.
Claims
Claims:
A recombinant transaminase polypeptide, wherein said polypeptide comprises amino acid substitutions selected from the group consisting of: i) Phe55 substituted with Cys or Met or any Aliphatic, Polar or non-polar or Aromatic amino acid; ii) Glu93 substituted with Pro or any Aliphatic, Acidic, or Polar or non-polar amino acid; iii) Glyl51 substituted with Ala, or any Aliphatic, non-polar, or Polar amino acid; iv) Thrl59 substituted with Ala, or any aliphatic or non-polar or polar amino acid; v) Glul66 substituted with Ala, or any Aliphatic, Acidic, Polar or non-polar, amino acid; vi) Val262 substituted with Met, or any Aliphatic, non-polar amino acid; vii) Ser298 substituted with Gly, or any Aliphatic or polar amino acid; viii) Ile427 substituted with Thr, or any Polar, or Aliphatic amino acid; ix) Val385 substituted with Leu, or He, or any non-polar or Aliphatic amino acid; x) Leu428 substituted with He, or any aliphatic or non-polar residue; xi) Leu58 substituted with Ser or Thr or any aliphatic polar amino acid; xii) Gly 154 substituted with Vai or Ala or He or Thr or any aliphatic amino acid; and xiii) Gly323 substituted with Vai or Ala or He or any aliphatic non-polar amino acid.
2. The recombinant transaminase polypeptide as claimed in claim 1, wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 to 45.
3. The recombinant transaminase polypeptide as claimed in claim 1, wherein said polypeptide is represented amino acid sequences selected from SEQ ID No. 1 to 45.
4. A polynucleotide encoding the recombinant transaminase polypeptide as claimed in claims 1-3, wherein said polynucleotide is selected from SEQ ID Nos. 46-56.
5. A fusion protein comprising a recombinant transaminase polypeptide as claimed in claims 1-3 and 6X His-tag.
6. An expression vector comprising the polynucleotide as claimed in claim 4.
7. A host comprising the expression vector as claimed in claim 6, wherein said host cell is a bacterial cell.
8. A process of preparing a compound of structural formula I,
(I) having the indicated stereochemical configuration at the stereogenic center markedwith an *; in an enantiomeric excess of at least 70 % over the opposite enantiomer, wherein
X is OR2;
R1 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-Ci-2 alkyl, carbocyclic, heterocyclic, optionally being unsubstituted or substituted with one to five fluorine, or any halogen; and
R2 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-C 1-2 alkyl, optionally being unsubstituted or substituted; wherein the process comprising the step of contacting a prochiral ketone compound of structural Formula (II):
X is OR2;
R1 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-Ci-2 alkyl, carbocyclic, heterocyclic, optionally being unsubstituted or substituted with one to five fluorine, or any halogen; and
R2 is alkyl, aryl, heteroaryl, benzyl, aryl-Ci-2 alkyl, heteroaryl-C 1-2 alkyl, optionally being unsubstituted or substituted; with the recombinant transaminase polypeptide as claimed in claim 1 ; in the presence of an amino group donor; and wherein the conversion is such that the product obtained has enantiomeric form “R”.
9. The process as claimed in claim 8, wherein R1 in the compound of structural formula I is a benzyl group, and wherein the phenyl group of benzyl is unsubstituted or substituted withone to five fluorines.
10. The process as claimed in claim 8, wherein the compound of formula I is (R)-3-amino- 4-(2,4,5-trifluorophenyl)butyric acid methyl ester.
11. The process as claimed in claim 8, wherein the compound of formula I is produced in the range of 60-100% enantiomeric excess.
12. The process as claimed in claim 8-11, wherein the amount of recombinant transaminases required for conversion of compound of structural formula II to compound of formula I is 1.5 mg/mL
13. The process as claimed in claim 8, wherein the rate of conversion of compound of formula II to compound of formula I ranges from 10% to 95%.
14. The process as claimed in claim 8, wherein the amino group donor is selected from o- Xylylenediamine or isopropyl amine.
15. The process as claimed in claim 8, wherein said process further comprises a step of protecting the amino group by an amino protecting group selected from formyl, acetyl, trifluoro acetyl, benzyl, benzyloxy carbonyl ("CBZ"), tert-butoxy carbonyl ("BOC"), trimethylsilyl ("TMS"), 2- trimethylsilyl-ethanesulfonyl (“SES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), and nitro-veratryl oxy carbonyl (“NVOC”).
16. The process as claimed in claim 15, wherein the amino protecting group is a BOC protecting group.
17. The process as claimed in claim 16, wherein the Boc protected chiral amine is (R)-3-(tert- Butoxycarbonylamino)-4-(2,4,5-trifluorophenyl)butanoicacid.
18. The process as claimed in claim 8, wherein the organic solvent is selected from dimethylsulfoxide (DMSO), Dimethylformamide (DMF), methyl tert-butyl ether (MTBE), isopropyl acetate, methanol, ethanol or propanol.
19. The process as claimed in claim 18, wherein the solvent is a mixture of water and dimethylsulfoxide (DMSO). 0. The process as claimed in claim 8, wherein the prochiral ketone of structural formula II is 3-oxo-4-(2,4,5-trifluorophenyl)butyric acid methyl ester. 1. The process as claimed in claim 8, wherein the process of preparing the compound of formula I further comprises the step of reacting the compound of formula I with , 3- (trifluoromethyl)-5,6,7,8-tetrahydro[l,2,4]triazolo[4,3-a]pyrazine, 3-(trifluoromethyl)- 6,8-dihydro-5/Z-imidazo[l,5-a]pyrazine, and 3-[(2-methylpropan-2- yl)oxymethyl]piperazin-2-one to make Sitagliptin, Retagliptin, and Evogliptin, respectively.
A method of designing the recombinant transaminase polypeptide as claimed in claim 1, wherein said method is performed in silico using QZyme Workbench™ and comprising the steps of
- structural refinement and modelling;
- ligand docking;
- conformational sampling;
- estimating substrate binding affinity;
- modelling catalytic reaction;
- identifying mutable hotspots and further optimizing the hotspot, and wherein said method provides an efficient recombinant transaminase polypeptide as claimed in claims 1-3, having increased catalytic activity. Use of the recombinant transaminase polypeptide as claimed in claims 1 to 3, in manufacturing of chiral amines, wherein said chiral amines are essential intermediates in production of pharmaceutical drugs selected from Sitagliptin, Evogliptin and Retagliptin, which treats diabetes mellitus type 2. A method of treating diabetes mellitus type 2, wherein said method comprising administering a subject a drug manufactured using intermediates obtained from the process as claimed in claims 8-21.
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| US20160304843A1 (en) * | 2015-04-16 | 2016-10-20 | Hoffmann-La Roche Inc. | Mutant transaminases as well as methods and uses relating thereto |
| CN107365809A (en) * | 2016-05-13 | 2017-11-21 | 上海朴颐化学科技有限公司 | A kind of method of transaminase method synthesis (R)-N-BOC-3- amino -4- (2,4,5- trifluorophenyls) butyric acid |
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| US20160304843A1 (en) * | 2015-04-16 | 2016-10-20 | Hoffmann-La Roche Inc. | Mutant transaminases as well as methods and uses relating thereto |
| CN107365809A (en) * | 2016-05-13 | 2017-11-21 | 上海朴颐化学科技有限公司 | A kind of method of transaminase method synthesis (R)-N-BOC-3- amino -4- (2,4,5- trifluorophenyls) butyric acid |
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| Title |
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| VOSS MORITZ, DAS DEVASHISH, GENZ MAIKA, KUMAR ANURAG, KULKARNI NAVEEN, KUSTOSZ JAKUB, KUMAR PRAVIN, BORNSCHEUER UWE T., HÖHNE MATT: "In Silico Based Engineering Approach to Improve Transaminases for the Conversion of Bulky Substrates", ACS CATALYSIS, AMERICAN CHEMICAL SOCIETY, US, vol. 8, no. 12, 7 December 2018 (2018-12-07), US , pages 11524 - 11533, XP055965098, ISSN: 2155-5435, DOI: 10.1021/acscatal.8b03900 * |
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