METHOD FOR PREPARATION OF PRIMARY AMINE COMPOUNDS

Abstract

The present invention relates to an enzyme-catalyzed enantioselective method for preparing primary amines from the corresponding imines by using imine reductase enzymes.

Claims

1. A method of preparing a primary amine compound of general formula (IB) ##STR00037## wherein R.sup.1 and R.sup.2 are independently selected from a hydrogen atom, and optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, thiocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; and optionally R.sup.1 and R.sup.2 are linked to form a 3-membered to 10-membered ring; comprising the step of: A1) Providing an imine compound of general formula (IIA) ##STR00038## wherein R.sup.1 and R.sup.2 have the meanings as defined above; and B1) Reacting imine compound of general formula (IIA) with a polypeptide having the enzymatic activity of an imine reductase in the presence of a cofactor, to afford primary amine of general formula (IB), wherein the polypeptide comprises a conserved amino acid as set forth in SEQ ID NO: 610.

2. The method of claim 1, wherein R.sup.1 represents —CH.sub.3, —COOR.sup.5, —COSR.sup.5, —CSSR.sup.5, —CONHR.sup.5, —CONR.sup.5R.sup.6, —CH═CH—COOR.sup.5, —CH═CH—COSR.sup.5, —CH═CH—CSSR.sup.5, —CH═CH—CONHR.sup.5, —CH═CH—CONR.sup.5R.sup.6, —C≡C—COOR.sup.5, —C≡C—COSR.sup.5, —C≡C—CSSR.sup.5, —C≡C—CONHR.sup.5 or —C≡C—CONR.sup.5R.sup.6; R.sup.2 represents —H, —X.sup.1, —CH.sub.3, —CF.sub.3, -Ph, —CH.sub.2Y.sup.1, —CH(Y.sup.1)Y.sup.2, cyclo-C.sub.3H.sub.5, cyclo-C.sub.4H.sub.7, cyclo-C.sub.5H.sub.9, cyclo-C.sub.6H.sub.11, cyclo-C.sub.7H.sub.13, cyclo-C.sub.8H.sub.15, —CH(OH)X.sup.1, —C(O)Y.sup.1, —CH(X.sup.1)X.sup.2, —CH.sub.2CH.sub.2X.sup.1, —(CH.sub.2).sub.3X.sup.1, —CH.sub.2C(O)X.sup.1 or —C(O)CH.sub.2X.sup.1 X.sup.1 and X.sup.2 are independently selected from —CN, —COOR.sup.7, —COOR.sup.8, —COSR.sup.7, —COSR.sup.8, —CSSR.sup.7, —CSSR.sup.8, —CONHR.sup.7, —CONHR.sup.8, —CONR.sup.7R.sup.9 or —CONR.sup.8R.sup.10, Y.sup.1, Y.sup.2 and Y.sup.3 are independently selected from —X.sup.1, —X.sup.2, —R.sup.13, —R.sup.14, or —R.sup.15, R.sup.5-R.sup.15 represent independently of each other —H, cyclo-C.sub.3H.sub.5, cyclo-C.sub.4H.sub.7, cyclo-C.sub.5H.sub.9, cyclo-C.sub.6H.sub.11, cyclo-C.sub.7H.sub.13, cyclo-C.sub.8H.sub.15, -Ph, —CH.sub.2-Ph, —CPh.sub.3, —CH.sub.3, —C.sub.2H.sub.5, —C.sub.3H.sub.7, —CH(CH.sub.3).sub.2, —C.sub.4H.sub.9, —CH.sub.2—CH(CH.sub.3).sub.2, —CH(CH.sub.3)—C.sub.2H.sub.5, —C(CH.sub.3).sub.3, —C.sub.5H.sub.11, —CH(CH.sub.3)—C.sub.3H.sub.7, —CH.sub.2—CH(CH.sub.3)—C.sub.2H.sub.5, —CH(CH.sub.3)—CH(CH.sub.3).sub.2, —C(CH.sub.3).sub.2—C.sub.2H.sub.5, —CH.sub.2—C(CH.sub.3).sub.3, —CH(C.sub.2H.sub.5).sub.2, —C.sub.2H.sub.4—CH(CH.sub.3).sub.2, —C.sub.6H.sub.13, —C.sub.3H.sub.6—CH(CH.sub.3).sub.2, —C.sub.2H.sub.4—CH(CH.sub.3)—C.sub.2H.sub.5, —CH(CH.sub.3)—C.sub.4H.sub.9, —CH.sub.2—CH(CH.sub.3)—C.sub.3H.sub.7, —CH(CH.sub.3)—CH.sub.2—CH(CH.sub.3).sub.2, —CH(CH.sub.3)—CH(CH.sub.3)—C.sub.2H.sub.5, —CH.sub.2—CH(CH.sub.3)—CH(CH.sub.3).sub.2, —CH.sub.2—C(CH.sub.3).sub.2—C.sub.2H.sub.5, —C(CH.sub.3).sub.2—C.sub.3H.sub.7, —C(CH.sub.3).sub.2—CH(CH.sub.3).sub.2, —C.sub.2H.sub.4—C(CH.sub.3).sub.3, —CH(CH.sub.3)—C(CH.sub.3).sub.3, —CH═CH.sub.2, —CH.sub.2—CH═CH.sub.2, —C(CH.sub.3)═CH.sub.2, —CH═CH—CH.sub.3, —C.sub.2H.sub.4—CH═CH.sub.2, —C.sub.7H.sub.15, —C.sub.8H.sub.17, —CH.sub.2—CH═CH—CH.sub.3, —CH═CH—C.sub.2H.sub.5, —CH.sub.2—C(CH.sub.3)═CH.sub.2, —CH(CH.sub.3)—CH═CH, —CH═C(CH.sub.3).sub.2, —C(CH.sub.3)═CH—CH.sub.3, —CH═CH—CH═CH.sub.2, —C.sub.3H.sub.6—CH═CH.sub.2, —C.sub.2H.sub.4—CH═CH—CH.sub.3, —CH.sub.2—CH═CH—C.sub.2H.sub.5, —CH═CH—C.sub.3H.sub.7, —CH.sub.2—CH═CH—CH═CH.sub.2, —CH═CH—CH═CH—CH.sub.3, —CH.sub.2NH.sub.2, —CH.sub.2OH, —CH.sub.2SH, —CH.sub.2—CH.sub.2NH.sub.2, —CH.sub.2—CH.sub.2SH, —C.sub.6H.sub.4—OCH.sub.3, —C.sub.6H.sub.4—OH, —CH.sub.2—CH.sub.2—OCH.sub.3, —CH.sub.2—CH.sub.2OH, —CH.sub.2—OCH.sub.3, —CH.sub.2—C.sub.6H.sub.4—OCH.sub.3, —CH.sub.2—C.sub.6H.sub.4—OH.

3. The method of claim 1, wherein R.sup.1 represents —COOH; and R.sup.2 is selected from —CH.sub.3, —COOH, —CH.sub.2CH.sub.3, —(CH.sub.2).sub.2CH.sub.3, —(CH.sub.2).sub.3CH.sub.3, —CH.sub.2COOH, —CH.sub.2CONH.sub.2, —CH.sub.2CH.sub.2OH, —CH.sub.2CH.sub.2SH, —CH(CH.sub.3)COOH, —(CH.sub.2).sub.2COOH or —(CH.sub.2).sub.2CONH.sub.2.

4. The method of claim 1, wherein the cofactor is NADH.

5. The method of claim 1, wherein the (R)-isomer of the primary amine of general formula (IB) is obtained.

6. The method of claim 5, wherein R.sup.1 represents —COOH, R.sup.2 represents —CHZ.sup.1Z.sup.2, and wherein Z.sup.1 represents —H or —CH.sub.3 and Z.sup.2 represents —H, —CH.sub.3, —CH.sub.2CH.sub.3, —(CH.sub.2).sub.2CH.sub.3, —COOH, —CONH.sub.2, —CH.sub.2OH, —CH.sub.2SH, —CH.sub.2COOH or —CH.sub.2CONH.sub.2.

7. The method of claim 1, wherein the imine compound of general formula (IIA) is provided by reacting a ketone or an aldehyde compound with ammonia or an ammonium salt.

8. The method of claim 7 comprising the steps of: A2) Providing a carbonyl compound of general formula (III) ##STR00039## and ammonia or an ammonium salt in order to form the primary imine of general formula (IIA); B2) Reacting the formed primary imine of general formula (IIA) with a polypeptide having the enzymatic activity of an imine reductase and a cofactor to afford the primary amine of general formula (IB), wherein the polypeptide comprises a conserved amino acid sequence as set forth in SEQ ID NO: 610.

9. The method of claim 6, wherein the imine compound of general formula (IIA) is provided by reacting an amino alcohol compound of general formula (V) with a β-hydroxyaspartate dehydratase ##STR00040## wherein R.sup.1 represents —COOH, R.sup.2* represents —C(OH)Z.sup.1Z.sup.2 and Z.sup.1 and Z.sup.2 have the meanings as defined in claim 6.

10. The method of claim 9, wherein the β-hydroxyaspartate dehydratase comprises an amino acid sequence of at least 80% sequence identity to SEQ ID NO: 602.

11. The method of claim 1, wherein the polypeptide having the enzymatic activity of an imine reductase comprises an amino acid of at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 300-598.

12. The method of claim 1, wherein the polypeptide having the enzymatic activity of an imine reductase comprises an amino acid sequence selected from SEQ ID NO: 300-598.

13. The method of claim 1, wherein the polypeptide having the enzymatic activity of an imine reductase comprises an amino acid sequence selected from SEQ ID NO: 300, 306, 321, 324, 325, 338, 346, 357, 374, 422, 434 and 459.

14. The method of claim 1, wherein the polypeptide having the enzymatic activity of an imine reductase comprises an amino acid sequence as set forth in SEQ ID NO: 434.

15. The method of claim 1, wherein R.sup.1 represents —COOH and R.sup.2 represents —CH.sub.2COOH.

Description

DESCRIPTION OF THE FIGURES

[0446] FIG. 1: illustrates the reaction sequence of the β-hydroxyaspartate pathway (BHAP) in Paracoccus denitrificans for the conversion of glyoxylate via the unstable iminosuccinate (shown in brackets) into oxaloacetate. The italic numbers represent the enzyme catalyzing the respective reaction: 1: aspartate-glyoxylate transaminase; 2: (2R,3S)-β-hydroxyaspartate aldolase (BHAA); 3: (2R,3S)-β-hydroxyaspartate dehydratase (BHAD) and 4: iminosuccinate reductase (ISRed). The net equation of the pathway is shown below the scheme.

[0447] FIG. 2: shows different reactions for the preparation of amines. FIG. 2A refers to the reductive amination of a ketone or aldehyde III and a secondary amine IV in order to obtain a tertiary amine I; FIG. 2B shows the reduction of a secondary imine compound II to a secondary amine compound IA; and FIG. 2C is directed to the inventive reduction of a primary imine compound IIA to a primary amine compound IB. The asterisks in formulae IA and IB indicate that a stereocenter is formed at the carbon atom attached to the amine nitrogen atom in case a chiral or pro-chiral imine compound was used.

[0448] FIG. 3: LC-MS analysis of monodeuterated L-aspartate formed of glycine and glyoxylate by the BHAA and BHAD enzymes in the presence of varying amounts of NaCNBH.sub.3 in D.sub.2O according to Example 3.

[0449] FIG. 4: LC-MS analysis of L-aspartate and (2R,3S)-β-hydroxyaspartate formed of glycine and glyoxylate by the BHAA, BHAD and Red enzymes according to Example 3.

[0450] FIG. 5: LC-MS-based analysis of the reactions catalyzed by β-hydroxyaspartate dehydratase (BHAD) and iminosuccinate reductase (ISRed). An overview of the relevant reactions is given in (A). To demonstrate that BHAD produces iminosuccinate, (2R,3S)-β-hydroxyaspartate (light grey) is incubated in .sup.2H.sub.2O with BHAD and with/without NaBH.sub.3CN to yield monodeuterated aspartate (medium grey; B) or oxaloacetate (white; C). To demonstrate that ISRed reduces iminosuccinate to aspartate, (2R,3S)-β-hydroxyaspartate is incubated with BHAD and with/without iminosuccinate reductase to yield aspartate (dark grey; D) or oxaloacetate (white; E). n=3, error bars depict standard deviation.

[0451] FIG. 6: Michaelis-Menten kinetics for the Red enzyme, with iminosuccinate synthesized in situ from BHA and varying amounts of the BHAD enzyme. The curve was fitted in GraphPad Prism 7. The v.sub.max is 270±10 U mg.sup.−1 (n=1).

[0452] FIG. 7: shows the crystal structure of the iminosuccinate reductase (ISRed). a, Cartoon representation of the iminosuccinate reductase homodimer (PDB ID 6QKH) with superimposed protein surface (side view—left panel, top view—right panel). b, Superimposition of monomers of iminosuccinate reductase and L-alanine dehydrogenase (PDB ID 1OMO) with bound NAD.sup.+. RMS of 1.287 Å over 241 Cα-atoms. c, Superimposition of the active sites of iminosuccinate reductase and L-alanine dehydrogenase. Although the binding of the nicotine amide moiety appears to be similar in both cases, the residues lining the active site from above differ substantially. These residues are located in the β-strands which also form the dimerization interface (see a for comparison) and are likely to be involved in the binding and orientation of the respective substrates above the nicotine amide moiety.

[0453] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples, which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those skilled 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.

[0454] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

EXAMPLES

Abbreviations and Acronyms

[0455] IRed imine reductase
ISRed iminosuccinate reductase
BHA (2R,3S)-β-hydroxyaspartate
BHAA (2R,3S)-β-hydroxyaspartate aldolase
BHAD (2R,3S)-β-hydroxyaspartate dehydratase
DNA desoxyribo nucleic acid

Chemicals & Reagents

[0456] Unless otherwise stated, all chemicals and reagents were acquired from Sigma-Aldrich, and were of the highest purity available.

Example 1: Construction of Expression Vectors for Heterologous Expression of the Imine Reductase, β-Hydroxyaspartate Aldolase and β-Hydroxyaspartate Dehydratase Polypeptide

[0457] The gene encoding for the imine reductase enzyme from Paracoccus denitrificans DSM 413 (IRed; nucleic acid sequence shown in SEQ ID NO: 135; amino acid sequence shown in SEQ ID NO: 434) was cloned into the standard expression vector pET16b (commercially available from Merck Millipore). To this end, the Red gene was amplified from genomic DNA of Paracoccus denitrificans DSM 413 with the primers

TABLE-US-00003 (SEQ ID NO: 603) 5′-GACGCCTCATATGCTCGTCGTCGCCGAAAAG-3′ (SEQ ID NO: 604) 5′-GCCACTCCTCGAGTCAGATCTCGACCTCTTG-3′ 

[0458] The resulting PCR product was digested with the endonucleases NdeI and XhoI and ligated into the expression vector pET16b to create a vector for heterologous expression of Red.

[0459] The gene encoding for the β-hydroxyaspartate aldolase enzyme from Paracoccus denitrificans DSM 413 (BHAA; nucleic acid sequence shown in SEQ ID NO: 599; amino acid sequence shown in SEQ ID NO: 600) was cloned into the standard expression vector pET16b (commercially available from Merck Millipore). To this end, the BHAA gene was amplified from genomic DNA of Paracoccus denitrificans DSM 413 with the primers

TABLE-US-00004 (SEQ ID NO: 605) 5′-GACGCCGCATATGAACGCGAAAACGGATTTC-3′ (SEQ ID NO: 606) 5′-GACACCTGGATCCTCAGTAGCCCTTTCCG-3′ 

[0460] The resulting PCR product was digested with the endonucleases NdeI and BamHI and ligated into the expression vector pET16b to create a vector for heterologous expression of BHAA.

[0461] The gene encoding for the β-hydroxyaspartate dehydratase enzyme from Paracoccus denitrificans DSM 413 (BHAD; nucleic acid sequence shown in SEQ ID NO: 601; amino acid sequence shown in SEQ ID NO: 602) was cloned into the standard expression vector pET16b (commercially available from Merck Millipore). To this end, the BHAD gene was amplified from genomic DNA of Paracoccus denitrificans DSM 413 with the primers

TABLE-US-00005 (SEQ ID NO: 607) 5′-GACGCTGCATATGTATATCCCGACCTATGAG-3′ (SEQ ID NO: 608) 5′-GACACTCGGATCCTCAGTTCCACGGCAGCTTG-3′ 

[0462] The resulting PCR product was digested with the endonucleases NdeI and BamHI and ligated into the expression vector pET16b to create a vector for heterologous expression of BHAD.

Example 2: Heterologous Expression and Purification of Recombinant Proteins

[0463] For the heterologous overexpression of the Red, BHAA and BHAD enzymes, respectively, the corresponding plasmid encoding the respective enzyme was first transformed into chemically competent E. coli BL21 AI cells. The cells transformed with the respective plasmid encoding one of said enzymes were then grown on LB agar plates containing 100 μg mL.sup.−1 ampicillin at 37° C. overnight. A starter culture in selective LB medium was then inoculated from a single colony on the next day and left to grow overnight at 37° C. in a shaking incubator. The starter culture was then used on the next day to inoculate an expression culture in selective TB medium in a 1:100 dilution. The expression culture was grown at 37° C. in a shaking incubator to an OD.sub.600 of 0.5 to 0.7, induced with 0.5 mM IPTG and 0.2% L-arabinose and grown overnight at 18° C. in a shaking incubator.

[0464] Cells were harvested at 6,000×g for 15 min and cell pellets were stored at −20° C. until purification of enzymes. Cell pellets were resuspended in two-fold volume of buffer A (300 mM NaCl, 25 mM Tris pH 8.0, 15 mM imidazole, 1 mM β-mercaptoethanol, 0.1 mM MgCl.sub.2, 0.01 mM pyridoxalphosphate (PLP), and one tablet of SIGMAFAST™ protease inhibitor cocktail, EDTA-free (Sigma-Aldrich) per L). The cell suspension was treated with a sonicator in order to lyse the cells and centrifuged at 50,000×g and 4° C. for 1 h. The supernatant was loaded onto 1 ml Protino® Ni-NTA Agarose (Macherey-Nagel) in a gravity column, which had previously been equilibrated with 5 column volumes of buffer A. The column was washed with 20 column volumes of buffer A and 5 column volumes of 85% buffer A and 15% buffer B and the respective protein was eluted with buffer B (buffer A, but with 500 mM imidazole). The eluate was desalted using PD-10 desalting columns (GE Healthcare) and buffer C (100 mM NaCl, 25 mM Tris pH 8.0, 1 mM MgCl.sub.2, 0.01 mM PLP, 0.1 mM DTT). The respective purified enzymes were stored at −20° C. in buffer C containing 50% glycerol.

Example 3: Enzyme Assays

[0465] The enzyme assay to generate iminosuccinate from glyoxylate and glycine (catalyzed by the BHAA and BHAD enzymes) and further chemical reduction of iminosuccinate to aspartate with the reducing agent NaCNBH.sub.3 was performed at 30° C. in a total volume of 600 μl. The reaction mixture contained 50 mM Tris pH 7.5, 1 mM glycine, 1 mM glyoxylate, 0.1 mM PLP, 1 mM MgCl.sub.2, 60 μg BHAA enzyme, 5.4 μg BHAD enzyme and varying amounts of NaCNBH.sub.3 (0, 1 or 5 mM, respectively). The reaction was carried out in deuterated water (D.sub.2O). 180 μL aliquots were taken at time points 0, 1 and 3 minute(s) and the reaction was immediately stopped by addition of formic acid (4% final concentration). The samples were centrifuged at 17,000×g and 4° C. for 15 min and the supernatant diluted 1:4 in double-distilled water for LC-MS analysis (see FIG. 3).

[0466] The enzyme assay to generate aspartate from glyoxylate and glycine (catalyzed by the BHAA, BHAD and ISRed enzymes) was performed at 30° C. in a total volume of 1 ml. The reaction mixture contained 50 mM MOPS/KOH pH 7.5, 1 mM glycine, 1 mM glyoxylate, 0.4 mM NADH, 0.1 mM PLP, 1 mM MgCl.sub.2, 100 μg BHAA enzyme, 9 ug BHAD enzyme and 1 μg ISRed enzyme. 180 μL aliquots were taken at time points 0, 1, 3, 5 and 10 minutes and the reaction was immediately stopped by formic acid (4% final concentration). The samples were centrifuged at 17,000×g and 4° C. for 15 min and the supernatant diluted 1:4 in double-distilled water for LC-MS analysis (see FIG. 4).

[0467] Thus, when incubating glyoxylate, glycine and the required cofactors together with the BHAA, BHAD and ISRed enzymes, increasing amounts of aspartate are formed over the course of 10 min.

[0468] The LC-MS measurements were done using an Agilent 6550 iFunnel Q-TOF LC-MS system equipped with an electrospray ionization source set to negative ionization mode. Liquid chromatography (LC) was carried out as follows: The analytes were separated on an aminopropyl column (30 mm×2 mm, particle size 3 μm, 100 Å, Luna NH.sub.2, Phenomenex) using a mobile phase system comprised of 95:5 20 mM ammonium acetate pH 9.3 (adjusted with ammonium hydroxide to a final concentration of approximately 10 mM)/acetonitrile (A) and acetonitrile (B). Chromatographic separation was carried out using the following gradient condition at a flow rate of 250 μl min.sup.−1: 0 min 85% B; 3.5 min 0% B, 7 min, 0% B, 7.5 min 85% B, 8 min 85% B. Column oven and autosampler temperature were maintained at 15° C. The ESI source was set to the following parameters: Capillary voltage was set at 3.5 kV and nitrogen gas was used as nebulizing (20 psig), drying (13 l/min, 225 C) and sheath gas (12 l/min, 400° C.). The QTOF mass detector was calibrated prior to measurement using an ESI-L Low Concentration Tuning Mix (Agilent) with residuals and corrected residuals less than 2 ppm and 1 ppm respectively. MS data were acquired with a scan range of 50-600 m/z. Autorecalibration was carried out using 113 m/z as reference mass. Subsequent peak integration of all analytes was performed using the eMZed software (Kiefer et al. Bioinformatics, 2013, 29(7), 963-964) (see FIG. 5).

[0469] The enzyme assay to generate aspartate from β-hydroxyaspartate (catalyzed by the BHAD and ISRed enzymes) was performed at 30° C. in a total volume of 300 μl. The reaction mixture contained 100 mM potassium phosphate pH 7.5, 1 mM BHA, 0.2 mM NADH, 0.57, 2.85, 5.7 or 11.4 μg BHAD enzyme and 0.57 μg ISRed enzyme. The oxidation of NADH was followed at 340 nm on a Cary 60 UV-Vis photospectrometer (Agilent) in quartz cuvettes with a pathlength of 10 mm (Hellma Analytics). BHA (=(2R,3S)-β-hydroxyaspartate) was custom-synthesized by the company NewChem (Newcastle upon Tyne, United Kingdom), and was determined to be >95% pure by NMR analysis. Therefore, the ISRed enzyme catalyzes the conversion of iminosuccinate into L-aspartate.

[0470] The enzyme assay to determine the initial reaction velocity of the ISRed enzymes was performed by incubating BHA and the required cofactor NADH together with a fixed amount of the ISRed enzyme and varying amounts of the BHAD enzyme. The initial velocity depends on the amount of the BHAD enzyme present in the reaction mixture, and therefore on the amount of iminosuccinate that is produced by the BHAD enzyme and is available to the ISRed enzyme, as illustrated in FIG. 6. The initial reaction velocity of the ISRed enzyme approaches a maximum in a Michaelis-Menten kinetic fit. The preliminary v.sub.max of the ISRed enzyme determined by this method is 270±10 U mg.sup.−1.

Example 4: Crystallization and Structure Determination of ISRed

[0471] The sitting-drop vapor-diffusion method was used for crystallization at 16° C. Purified ISRed (5 mg ml.sup.−1) was mixed in a 1:1 ratio with solution B containing 20% PEG 3350, 0.06 M BIS-TRIS propane, and 0.04 M citric acid, pH 6.4 (final drop volume 4 μL). Reservoirs were filled with 114 μL of solution B. Crystals appeared within 12 days. Crystals were briefly soaked in mother liquor supplemented with 12 mM NAD.sup.+ and 40% MPD (2-Methyl-2,4-pentanediol) for cryoprotection before freezing in liquid nitrogen.

[0472] X-ray diffraction data were collected at the beamlines ID29 and ID30B of the ESRF (Grenoble, France). The data was processed with the XDS (Kabsch, W. (2010). “Xds.” Acta Crystallogr D Biol Crystallogr 66(Pt 2): 125-132). (BUILT 20180126) and CCP4 7.0 software packages (Winn et al. “Overview of the CCP4 suite and current developments.” Acta Crystallogr D Biol Crystallogr 67(Pt 4): 235-242). The structures were solved by molecular replacement. A homology model was made based on the structure of L-alanine dehydrogenase (PDB ID 1OMO) (Gallagher et al. “Structure of alanine dehydrogenase from Archaeoglobus: active site analysis and relation to bacterial cyclodeaminases and mammalian mu crystallin.” J Mol Biol 342(1): 119-130.) using Swiss-Model (Waterhouse et al. “SWISS-MODEL: homology modelling of protein structures and complexes.” Nucleic Acids Res 46(W1): W296-W303.). This homology model was then used as search model for the molecular replacement. The molecular replacement was carried out using Phaser of the Phenix software package (Adams et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution.” Acta Crystallogr D Biol Crystallogr 66(Pt 2): 213-221.) (version 1.14), built with Phenix.Autobuild, and refined with Phenix.Refine. Additional modeling, manual refining and ligand fitting was done in Coot (Emsley et al. “Coot: model-building tools for molecular graphics.” Acta Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1): 2126-2132.) (version 0.8.9). Final positional and B-factor refinements, as well as water-picking for the BHAA structure were performed using Phenix.Refine. The structure models for BHAA and ISRed were deposited at the Protein Bata Bank in Europe (PDBe) under the PDB ID 6QKB and 6QKH, respectively. FIG. 7 was made using Pymol 1.8.

[0473] A sequence listing is attached to this application comprising the sequences of the following table:

TABLE-US-00006 SEQ ID description  1-299 nucleic acids encoding polypeptides having the enzymatic activity of an imine reductase 300-598 polypeptides having the enzymatic activity of an imine reductase 599 beta-hydroxyaspartate aldolase 601 beta-hydroxyaspartate dehydratase 603 PCR Primer for imine reductase polypeptide encoding gene 604 PCR Primer for imine reductase polypeptide encoding gene 605 PCR primer for beta-hydroxyaspartate aldolase polypeptide encoding gene 606 PCR primer for beta-hydroxyaspartate aldolase polypeptide encoding gene 607 PCR primer for beta-hydroxyaspartate dehydratase polypeptide encoding gene 608 PCR primer for beta-hydroxyaspartate dehydratase polypeptide encoding gene 609 pET-16b expression vector 610, 611 Conserved amino acid sequence of the iminosuccinate reductases of SEQ ID NO: 300-598 612 Conserved amino acid sequence of the iminosuccinate reductases of SEQ ID NO: 300, 306, 321, 324, 325, 338, 346, 357, 374, 422, 434 and 459 613-616 Conserved amino acid sequence of the iminosuccinate reductases 617 Conserved amino acid sequence of the iminosuccinate reductases of SEQ ID NO 303, 309, 310, 318, 344, 345, 347, 362, 385, 386, 396, 397, 414, 432, 434, 446, 459, 470, 472, 491, 503, 509, 513, 524, 539, 564, and 576 618 Conserved amino acid sequence of the iminosuccinate reductases of SEQ ID NO 303, 309, 344, 345, 347, 385, 386, 396, 414, 432, 434, 446, 459, 470, 472, 503, 513, 539, 564, and 576. 619 Conserved amino acid sequence of the iminosuccinate reductases of SEQ ID NO 303, 344, 345, 347, 385, 386, 396, 414, 432, 434, 470, 472, 503, 539, and 576. 620 Conserved amino acid sequence of the iminosuccinate reductases of SEQ ID NO: 303, 344, 345, 385, 386, 396, 414, 432, 434, and 470. 621 Conserved amino acid sequence of the iminosuccinate reductases of SEQ ID NO 303, 345, 396, 432, and 434.