Revealing the Structural Basis of Promiscuous Amine Transaminase Activity

Abstract

One lock for different keys: A flexible arginine in the active site allows γ‐aminobutyrate:pyruvate transaminases to bind the chemically different substrates L‐alanine and γ‐aminobutyric acid. Moreover, a flexible arginine residue facilitates the promiscuous conversion of (S)‐amines and ketones. The degree of promiscuity can be related to distinct key amino acids lying at the surface of the active site.

A new target region for changing the substrate specificity of amine transaminases

“…ATAs transfer an amino group of a chiral amine compound to a ketone compound using the cofactor pyridoxal 5’-phosphate (PLP) (Supplementary Fig. 1a) with a high turnover rate, stable catalytic activity, broad substrate specificity and excellent stereoselectivity7, which is achieved by a proposed large-binding pocket (L pocket) and small-binding pocket (S pocket) in the substrate-binding site8910111213141516 (Supplementary Fig. 1b).…”

A subfamily of PLP-dependent enzymes specialized in handling terminal amines

“…Aminotransferase reactions, on the other hand, are intrinsically rather slow processes, owing to their complex ping-pong mechanism. 1 The expression "amine transaminases" is also used when describing ATs employed for bio-organic synthetic applications [14,24,25]. In the field, this tautological expression is used in a restricted sense, to indicate enzymes acting on amines that are neither terminal nor adjacent to a carboxylate.…”

“…Since in a large O-pocket the internal arginine can move relatively freely, it does not need to mask its positive charge, as it happens in the gateway system, to avoid clashes with the hydrophobic side chain of the amino substrate. Recently Steffen-Munsberg et al, based on the analysis of some ω-ATs, contrasted the mobility of the Arg residue in AT-I and AT-II enzymes [24]. According to this analysis, in AT-I the conserved Arg residue is quite packed within the active site, and it can only contact the α-carboxylic group of the substrate, while in AT-II enzymes the Arg is very flexible.…”

“…The O-pocket of α-KG-specific transaminases is smaller and more constrained than that of pyruvate-specific enzymes [24] (Fig. 4C).…”

Fluorescence‐Based Kinetic Assay for High‐Throughput Discovery and Engineering of Stereoselective ω‐Transaminases

“…[7] Particularly, thermostable enzymes are expected to offer several advantages over normal w-transaminase in biotechnology,s uch as maintaining high activity under continuous process operation, improving tolerance for organic co-solvent and reducing overall reactiont imes due to increasedc atalytic activity at higher reaction temperatures.B oth the discovery of novel w-transaminases and determination of their enantioselectivity, as well as the protein engineering of known w-transaminases to improve theirs ubstrate scope rely on effective methods to rapidlye valuate biocatalyst activity and stereoselectivity with high accuracy.A lthough X-ray protein crystal structures have been determined for both (R)-and (S)-selective transaminases [8][9][10][11] and although it couldb es hown that the enantioselectivity and substrate scope of novel transaminases can be predicted from specifics ignatures in their genetic sequence, [12] an ultimate typing of their enantiopreference and substrate scope requirese xperimental proof by an appropriate assay technology.…”