Insight into the dimer dissociation process of the Chromobacterium violaceum (S)-selective amine transaminase

Federica Ruggieri,Derek T. Logan,Shan Chen,Maria S. Humble,Jonatan C. Campillo-Brocal,Björn Walse,Per Berglund

doi:10.1038/s41598-019-53177-3

Abstract

One of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability. Our still limited understanding of the inactivation processes undermining the stability of wild-type transaminases represents an obstacle to improving their stability through enzyme engineering. In this paper we present a model describing the inactivation process of the well-characterized (S)-selective amine transaminase from Chromobacterium violaceum. The cornerstone of the model, supported by structural, computational, mutagenesis and biophysical data, is the central role of the catalytic lysine as a conformational switch. Upon breakage of the lysine-PLP Schiff base, the strain associated with the catalytically active lysine conformation is dissipated in a slow relaxation process capable of triggering the known structural rearrangements occurring in the holo-to-apo transition and ultimately promoting dimer dissociation. Due to the occurrence in the literature of similar PLP-dependent inactivation models valid for other non-transaminase enzymes belonging to the same fold-class, the role of the catalytic lysine as conformational switch might extend beyond the transaminase enzyme group and offer new insight to drive future non-trivial engineering strategies.

Highlights

One of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability
The transamination cycle catalyzed by Chromobacterium violaceum (S)-ATA (Cv-ATA) follows the general ping-pong bi-bi reaction mechanism accepted for transaminases[21], relying on the formation of a reactive Schiff base between the side chain of the catalytic lysine (K288) and the 4′ group of the cofactor PLP (Fig. 1, panel d)
The further participation of the K288 terminal amino group in catalysis as proton acceptor/donor (Supplementary, Scheme S1) requires its physical proximity to the 4′ group of the PLP ring. This proximity condition is not met in the apo-Cv-ATA conformation (PDB IDs 4A6R and 4BA4)[20,22], where the side chain of the catalytic lysine is oriented towards the back of the active site, away from the 4′ group of the cofactor PLP

Summary

Introduction

One of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability. One of the main factors limiting the implementation of transaminases on an industrial scale for the synthesis of chiral amines is their poor storage and operational stability[3,6] While other properties such as substrate scope, enantioselectivity and inhibition have been successfully altered through enzyme engineering in a number of instances[3], the improvement of the stability profile of transaminases has only gained momentum in the last couple of years. Computational and experimental studies show that the stabilization role of PLP is exerted primarily at the level of the catalytic lysine K288 and secondarily at the level of the PGBC This model unifies the available structural information and stability data and provides a new insight into the PLP-dependent dimer stability of Cv-ATA

Methods

Results

Conclusion