Characterization of a Putrescine Transaminase From Pseudomonas putida and its Application to the Synthesis of Benzylamine Derivatives

Abstract

The reductive amination of prochiral ketones using biocatalysts has been of great interest to the pharmaceutical industry in the last decade for integrating novel strategies in the production of chiral building blocks with the intent of minimizing impact on the environment. Amongst the enzymes able to catalyze the direct amination of prochiral ketones, pyridoxal 5′-phosphate (PLP) dependent ω-transaminases have shown great promise as versatile industrial biocatalysts with high selectivity, regioselectivity, and broad substrate scope. Herein the biochemical characterization of a putrescine transaminase from Pseudomonas putida (Pp-SpuC) was performed, which showed an optimum pH and temperature of 8.0 and 60°C, respectively. To gain further structural insight of this enzyme, we crystallized the protein in the apo form and determined the structure to 2.1 Å resolution which revealed a dimer that adopts a class I transaminase fold comparable to other class III transaminases. Furthermore we exploited its dual substrate recognition for biogenic diamines (i.e., cadaverine) and readily available monoamines (i.e., isopropylamine) for the synthesis of benzylamine derivatives with excellent product conversions and extremely broad substrate tolerance.

Development of Continuous Flow Systems to Access Secondary Amines Through Previously Incompatible Biocatalytic Cascades**

“…This approach was expanded to generate N ‐(2‐phenyl)ethyl cyclohexylamine 11 using another metagenomic RedAm (IR‐23), again only requiring two equivalents of amine with a system residence time of 24 mins (STY: 2.53 g L −1 h −1 ). A third approach was then used to generate N ‐benzylcyclohexylamine 12 with the transaminase from Pseudomonas putida ( Pp TA) [39] mediated transamination of benzaldehyde followed by reductive amination with cyclohexanone using IR‐79. Pleasingly 95 % conversion to 12 was achieved and maintained for four hours with a system residence time of 24 mins (STY: 2.24 g L −1 h −1 ).…”

Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin‐derived substrates

“…This indicates that (i) the endogenous ATAs lack activity towards VA or (ii) that the endogenous ATAs are expressed at an insufficient level to sustain cell growth under the applied conditions. To investigate which of the two alternatives was the case, we searched for putative native transaminases in P. putida KT2440 (taxid: 160488) by using BLASTp analysis (Dalal and Atri, 2014) with two ATAs as query sequences; Cv‐ATA from Chromobacterium violaceum (Kaulmann et al ., 2007; Du et al ., 2014) and Pp‐SpuC from P. putida (taxid: 303; Galman et al ., 2017; Galman et al ., 2018). Cv‐ATA (PDB id: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4BA5) was used as a query sequence since it has previously been found to catalyse the desired reaction (Kaulmann et al ., 2007), and the putrescine transaminase Pp‐SpuC (PDB id: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6HX9) was chosen because it has been shown to recognize a broad spectrum of aromatic compounds as substrate (Galman et al ., 2017; Galman et al ., 2018).…”

“…To investigate which of the two alternatives was the case, we searched for putative native transaminases in P. putida KT2440 (taxid: 160488) by using BLASTp analysis (Dalal and Atri, 2014) with two ATAs as query sequences; Cv‐ATA from Chromobacterium violaceum (Kaulmann et al ., 2007; Du et al ., 2014) and Pp‐SpuC from P. putida (taxid: 303; Galman et al ., 2017; Galman et al ., 2018). Cv‐ATA (PDB id: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4BA5) was used as a query sequence since it has previously been found to catalyse the desired reaction (Kaulmann et al ., 2007), and the putrescine transaminase Pp‐SpuC (PDB id: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6HX9) was chosen because it has been shown to recognize a broad spectrum of aromatic compounds as substrate (Galman et al ., 2017; Galman et al ., 2018). The top three ATAs displaying the lowest e‐value and highest sequence homology to both Pp‐SpuC and Cv‐ATA were chosen for further investigations and denoted here as Pp‐SpuC‐I (gene PP_2180), Pp‐SpuC‐II (gene PP_5182) and Pp‐ATA (gene PP_2588), respectively (Table S1).…”

“…S1 ).VA is not likely the natural substrate for the native ATAs presented here, although they have activity against VA in different levels. The Pp‐SpuC‐II, which is closely related to Pp‐SpuC from other P. putida strain (taxid: 303), is a putrescine transaminase that has preference for aliphatic diamines (Galman et al ., 2017 ; Galman et al ., 2018 ). Wu et al .…”

“…For this purpose, a novel growth‐based screening method suitable for high‐throughput evaluation of candidate genes was developed. Identified genes encoding the most active enzymes, putrescine transaminase Pp‐SpuC‐II (Galman et al ., 2018 ) and the well‐known ATA from Chromobacterium violaceum (Cv‐ATA; Kaulmann et al ., 2007 ) were overexpressed in the strain GN442ΔPP_2426 (García‐Hidalgo et al ., 2020 ). Furthermore, the effect of coexpressing Alanine dehydrogenase (AlaDH) from Bacillus subtilis for the regeneration of l ‐alanine was evaluated.…”