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Item Open Access Characterization and application of novel imine reductases(2016) Scheller, Philipp; Hauer, Bernhard (Prof. Dr.)Chiral amines are an ubiquitously distributed class of bioactive compounds, what turns them into preferred scaffolds for pharmaceuticals. The high chemical and enantiomerical purities required for such an application are ideally suited for biocatalysis as enzymatic methods routinely display high specificities. The established methods for chiral amine synthesis with lipases, ω-transaminases and amine oxidases, however have considerable limitations regarding their access to pharmaceutically relevant chiral secondary and tertiary amines. Recently the new enzyme class of imine reductases (IREDs) was described, offering an attractive extension to the currently used techniques as the preparation of imines by chemical methods in organic solvents is a well established and widely applicable method. As the number of IREDs known initially was limited to only three enzymes, this project started with a database search for the discovery of novel enzymes. For the first time it was shown that the IRED family is much larger than assumed and over 350 novel, putative IREDs were identified. A sequence analysis of the database members revealed (R)- type and (S)- type superfamilies and led after an update to the identification of IRED specific sequence motifs. These criteria allowed to define this new enzyme family on a sequence level and discriminate them from the closest related homologues. Based on the biochemical information about the three published IREDs and a conservation analysis of the database members, three new enzymes from Streptosporangium roseum DSM43021, Streptomyces turgidiscabies and Paenibacillus elgii were selected for characterization. The enzymes were shown to encode for functional IREDs with much higher activity than the previously known IREDs. By site directed mutagenesis the mechanism of the IREDs was probed and the importance of a conserved Tyr for catalysis of an (S)- type IRED shown, while the crucial role of the proposed Asp residue for catalysis in the (R)- type IREDs was questioned. The characterization of the new IREDs revealed their pH optima and confirmed the suspected dimerization. The thermostability of the IREDs was investigated and the selected (S)- type IRED identified as the most stable enzyme known to date. Further the activity in the presence of water miscible organic solvents was tested and high tolerance versus MeOH found. In biotransformations all IREDs showed high activity and a broad panel of cyclic imines was fully converted to piperidines and tetrahydroisoquinolines with enantioselectivities up to 99% ee. With purified IREDs kinetic constants for these substrates were recorded and their substrate preference investigated. This indicated a preference of the (S)- type IRED for more bulky substrates, compared to the (R)- type IREDs. After optimization of the reaction conditions, with purified IREDs also high activities and chemo- as well as enantioselectivities for very labile exocyclic imines were detected. The possibility to effectively reduce already low levels of such imines led to the application of one (R)- type IRED for the generation of novel C-N bonds by reductive aminations. The established methodology revealed the crucial influence, conditions that favor imine formation (high molar excess of the amine nucleophile and high pH) display on the conversion rates. Under optimized conditions, different carbonyls could efficiently be transformed with a variety of amines in the aqueous buffer system with moderate to good conversions into primary and secondary (chiral) amines with very high selectivities (ee up to 98%). Finally, an application of IREDs in cascade reactions to produce saturated N-heterocyclic compounds was envisioned. A microbial putrescine oxidase (PuO) was chosen to selectively oxidize polyamines to aminoaldehydes, thereby triggering their spontaneous cyclization to an imine. To target a broad range of heterocycles, PuO was characterized with a range of unnatural polyamines. The results indicated a narrow substrate scope and low activity for these compounds. To enhance the activity for such substrates directed evolution of PuO with epPCR was performed and led to the identification of a Glu residue, representing a hotspot for mutagenesis. This residue aligns to one of the multiple channels that lead into the deeply buried active site of PuO and it is located in the second shell around the active site. By site-saturation mutagenesis further mutants in the active site and this channel were generated and many mutants with smaller amino acids demonstrated the influence of this hotspot position to increase the activity of the enzyme. The best mutant exhibited considerably increased activities (up to 25-fold) for unnatural polyamines and also for natural polyamines the substrate spectrum was strongly shifted from putrescine towards longer polyamines like spermidine which is now transformed with a 10-fold increased catalytic efficiency (kcat/KM). The combination of both enzymes in purified form as well as in whole cells enabled the production of heterocyclic amines relying on the consecutive transformation of the substrate by both enzymes. While with the whole cell system only low amounts of the N-heterocyclic compounds were produced, the utilization of purified enzymes led in case of all three IREDs to high conversions of different polyamines into pyrrolines and piperidines.