Structure and Mutation of the Native Amine Dehydrogenase MATOUAmDH2

Abstract Native amine dehydrogenases (nat‐AmDHs) have recently emerged as a potentially valuable new reservoir of enzymes for the sustainable and selective synthesis of chiral amines, catalyzing the NAD(P)H‐dependent ammoniation of carbonyl compounds with high activity and selectivity. MATOUAmDH2, recently identified from the Marine Atlas of Tara Oceans Unigenes (MATOUv1) database of eukaryotic genes, displays exceptional catalytic performance against its best identified substrate, isobutyraldehyde, as well as having broader substrate scope than other nat‐AmDHs. In the interests of providing a platform for the rational engineering of this and other nat‐AmDHs, we have determined the structure of MATOUAmDH2 in complex with NADP+ and also with the cofactor and cyclohexylamine. Monomers within the structure are representative of more open and closed conformations of the enzyme and illustrate the profound changes undergone by nat‐AmDHs during the catalytic cycle. An alanine screen of active site residues revealed that M215A and L180A are more active than the wild‐type enzyme for the amination of cyclohexanone with ammonia and methylamine respectively; the latter suggests that AmDHs have the potential to be engineered for the improved production of secondary amines.


Expression and Purification of MATOUAmDH2 from pET22b
The MATOUAmDH2 gene had been cloned into a pET22b vector with a non-cleavable N-terminal histidine tag as described previously. [1] The recombinant plasmid was used to transform BL21(DE3) (New England BioLabs) cells and heat shocked at 42⁰C for 45 s. 1 mL of Luria Bertani (LB) medium was added and the suspension was incubated at 37⁰C for 1 h with shaking at 120 r.p.m. 100 μL of this culture were then added to LB agar plates containing 100 μg mL -1 ampicillin and these were incubated at 37⁰C overnight. Single colonies were selected and inoculated into 10 mL LB broth containing 100 μg mL -1 ampicillin. These cultures were incubated for 18 h at 120-180 r.p.m at 37⁰C with shaking and then transferred to 2 L flasks containing 1 L LB broth containing 100 μg mL -1 ampicillin. These cultures were incubated at 37⁰C until they reached an absorbance value of 0.6 at 600 nm, upon which they were induced with the addition of 1 mM isopropyl--D-thiogalactopyranoside (IPTG). Following induction, cells were left to grow at 16⁰C for 18 h with shaking at 180 r.p.m. Cells were then harvested using a Lynx6000 centrifuge (Thermo Scientific) at 9760 g for 20 min. The pellets were resuspended in 100 mL of a buffer containing 50 mM Tris-HCl, 300 mM NaCl and 20 mM imidazole at a pH of 7.1. Resuspended cells were disrupted using a cell disruptor homogenizer (Constant Systems Ltd) at a pressure of 26-27 kPsi.
Lysed cells were then centrifuged at 38,571 g for 40 min and the supernatant retained for enzyme purification.
The cell supernatant was filtered through a 0.45 μm filter before being loaded onto a 5 mL FF HisTrap column (GE Healthcare) pre-loaded with nickel sulfate.
MATOUAmDH2 was eluted using a low UV imidazole gradient formed from start and limit buffers of 20 mM to 500 mM respectively, each contained within a buffer of 50 mM Tris-HCl and 300 mM NaCl at pH 7.1. Fractions containing MATOUAmDH2, as determined by SDS-PAGE were concentrated to a volume of 2 mL using 10,000 MWCO concentrators before being filtered, as above, and loaded onto a Superdex gel filtration S200 16/60 column. Following elution, fractions were analysed using SDS-PAGE ( Figure S1).

Subcloning of MATOUAmDH2 gene in pETYSBLIC-3C
The gene encoding MATOUAmDH2 was subcloned into the pETYSBLIC-3C vector in order to explore an alternative construct for protein crystallization. The MATOUAmDH2 gene was amplified from the pET22b construct by PCR using the primers in Table S1.

GGAGAAGGCGCCTTTAAAGACGGCCATGAACGTACGC
Following the PCR, 20 units Dpn1 (1 µL) were added to the mixture, which was then made up to 50 µL with water and CutSmart buffer (NEB). The Dpn1 digestion reaction was incubated at 37⁰C for 20 min and then a heat inactivation step was performed at 80⁰C for 20 min. The reaction was run on an agarose gel and the band of a size corresponding to the MATOUAmDH2 gene (approx 1 kB) excised and the DNA purified. In-Fusion cloning (Takara Bio) was carried out using the protocol provided by the vendor using a 2:1 ratio of insert to vector. The In-Fusion reaction was incubated at 50⁰C for 15 min and then placed on ice. 2.5 µL of the reaction was then added to 25 µL of thawed Stellar competent cells and placed on ice for 30 min. The cells were then heat shocked at 42⁰C for 45 s and then the transformed cells placed on ice for 2 min. The total volume was brought to 500 µL with SOC media and shaken at 180 r.p.m at 37⁰C for 1 h. The culture was plated out onto agar containing kanamycin (30 μg mL -1 ) and incubated at 37⁰C overnight. Single colonies were selected and plasmids miniprepped. The successful cloning was first confirmed by a double restriction digest using NcoI and NdeI and then by DNA sequencing.

Expression and Purification of MATOUAmDH2 from pETYSBLIC-3C
MATOUAmDH2 in the PETYSBLIC3C vector was produced in an identical manner to that from the construct in pET22b, except that the antibiotic marker in transformation experiments and cell growths was kanamycin, rather than ampicillin, and was added in all cases to a final concentration of 30 μg mL -1 . Gel fractions, run as above, for the MATOUAmDH2 pETYSBLIC-3C construct are provided below in Figure S2.

For MATOU AmDH2 purified from pET22b expression
Fractions containing MATOUAmDH2, as determined by SDS-PAGE were pooled and concentrated to 10 mg mL -1 for crystallization experiments. Purified MATOUAmDH2 was complexed with 10 mM NADP + and subjected to crystallization trials using a range of commercially available screens in 96-well-plate sitting-drop format using a Mosquito robot (TTP LabTech). For crystallization drops a 1:1 ratio of protein solution (150 nL) to mother liquor (150 nL) was used. After initial trials, the best hits were obtained from

For MATOUAmDH2 purified from pETYBSLIC-3C
MATOUAmDH2 was concentrated to 10 mg mL -1 was mixed with 10 mM NADP + and 10 mM cyclohexylamine, after which the protein solution was incubated on ice for at least 30 min. Following screening against commercial screens using the Mosquito robot (TTP LabTech) in 96 well plates, the conditions were then optimised further in 48-well and 96-well plate formats (ratios as above). The best hits were obtained from 96-well optimisations in drops containing 0.2 M MgCl2, 0.1 M Bis-Tris pH 6.5 and 25% (w/v) PEG 3350. Crystals were fished from initial crystal screens and were flashcooled in liquid nitrogen with no additional cryoprotectant added. and their diffraction was tested using an in-house X-ray generator with a rotating anode (Rigaku).
Diffraction patterns were visualised using Advx (v.1.9.14) imaging and the best sent off to the Diamond Light Source, Oxford, UK on various beamlines.

Data Collection and Refinement
Data were collected on beamlines I03 and I04 at the Diamond Light Source and were processed and integrated using XDS [2] and scaled using SCALA [3] within the Xia2 [4] processing system. Data collection statistics can be found in Table S2. Crystals of MATOUAmDH2 from the pET22b construct were obtained in space group P212121 with four molecules in the asymmetric unit constituting two dimers. The structure was solved with MOLREP [5] using the structure of the amine dehydrogenase from Cystobacter fuscus (33% sequence identity, PDB code 6IAU) [6] as the molecular replacement model. The structure was solved using iterative cycles of the programmes COOT [7] and REFMAC5. [8] After building of the protein backbone, side chains and water molecules, residual density was present in the omit map in all four active sites.
This could be modelled as NADP + in each case. Crystals of MATOUAmDH2 from the pETYSBLIC3C construct were obtained in the P321 space group with one molecule in the asymmetric unit. This structure was solved using the MATOUAmDH2 structure as a model and built and refined as for the other structure. In this case the omit maps revealed clear density in the active site that could be modelled as NADP + and also the amination product cyclohexylamine. The Ramachandran plot for the NADP + complex revealed 98.4%, 1.3% and 0.3% of residues in highly preferred, preferred and outlier regions respectively. The corresponding figures for the NADP + -CHA complex were 98.7%, 1.0% and 0.3%. Refinement statistics for the structures can be found in Table   S2. Coordinates and structure factor files have been deposited in the Protein Data Bank (PDB) for the MATOUAmDH2 in complex with NADP + and with NADP + and cyclohexylamine with the accession codes 7ZBO and 7R09 respectively. map at a level of 3observed prior to ligand building and refinement. The cofactor atoms from the final structures have been added for clarity.

Site-Directed Mutagenesis
Mutants of MATOUAmDH2 were created using the pETYSBLIC-3C construct as the template and primers that were designed with alanine mutations at sites F143, L144, L169, L180, M215 and T312. The primers are listed in Table S3. MATOUAmDH2 muteins were expressed and purified as for the WT enzyme in both the pET22b and pETYSBLIC-3C constructs, described in protocols above.

Kinetic Measurements
The protocol closely followed the one describe by Caparco and co-workers. [

Biotransformations
The progress of amination reactions catalyzed by MATOUAmDH2 was monitored by

Modelling
Automated docking was performed using AUTODOCK VINA 1.1.2. [9] The monomer structure of MATOUAmDH2 from the P321 dataset was prepared using AUTODOCK utility scripts. Coordinates for iminium intermediate 16 were prepared using ACEDRG [10] in the ccp4 suite.The active site of MATOUAmDH2 was contained in a grid of 32 x 32 x 32 Å respectively with 0.375 Å spacing, centred around the catalytic centre which was generated using AutoGrid in the AUTODOCK Tools interface. The number of runs for genetic algorithm was set to 10 and the rest of the docking parameters were set to default parameters. The dockings were performed by VINA, therefore the posed dockings were below 2 Å rmsd. The results generated by VINA were visualised in AUTODOCK Tools 1.5.6 where the ligand conformations were assessed upon lowest VINA energy, and yielded the result in Figure 6 as the top pose.