Bioprospecting Reveals Class III ω-Transaminases Converting Bulky Ketones and Environmentally Relevant Polyamines

Abstract

Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (R)- and (S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways.

Transaminases for industrial biocatalysis: novel enzyme discovery

“…Interestingly, however, 4 of these enzymes (TR 2 , TR 6 , TR 9 , and TR 10 ) were able to accept both the S-and Renantiomers of 2-aminononane. Unlike the other Class III TAms uncovered by this screen, these enzymes all had a large pocket volume, a hairpin region close to the conserved arginine residue at position 414 and an outward orientation of the Arg414 residue itself, although the authors postulate that other factors may be at play in determining the ability of these enzymes to accept both S-and R-substrates (Coscolín et al 2019).…”

“…To date, functional metagenomic studies for TAms are scarce but have involved the generation of fosmid libraries for novel enzyme discovery. In one such approach, metagenomic fosmid libraries from 28 geographically distinct environments were produced and screened using wellestablished agar-based colorimetric assays (Green et al 2014;Baud et al 2015) to show clones harboring relevant TAm-encoding sequences (Coscolín et al 2019). Analysis of candidates at protein sequence level revealed large divergence among the Class III TAms identified, with no particular type of protein dominant.…”

“…As with other discovery methodologies discussed, functional metagenomics is not without limitations. While colorimetric screens such as those employed by Coscolín et al (2019) and Pawar et al (2018) increase screening efficiency considerably, expression and screening of clones is not trivial and can be time consuming and resource heavy. Furthermore, not all enzymes can be easily expressed in established E. coli-based systems, such as those from extremophiles, whose characteristics can be very desirable.…”

Biocatalysis

“…2c). Functionalbased screening can incorporate the use of colorimetric assays [30][31][32] , mechanism-based probes 33 and/or droplet microfluidics 34 . Researchers are looking for enzymes in these libraries that demonstrate catalysis of the desired…”

Genome mining and characterisation of a novel transaminase with remote stereoselectivity

“…Liquid and solid-phase screening technologies have emerged in recent years enabling greater access to evolved TA protein variants. These screening platforms enable access to natural biodiversity 75 , as well as evolved variants 76 . However, notwithstanding these advances, the industrial application of these novel TAs remains limited by factors such as disfavoured reaction equilibrium, poor substrate scope, and product inhibition 77 .…”