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

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.

Structural studies reveal flexible roof of active site responsible for ω-transaminase CrmG overcoming by-product inhibition

“…Other than the roof of the active site, the structural rearrangement of the interfacial loop, outer loop, and N terminal on the active site cavity was also observed in Cv-ωTA upon PLP binding 22,24 . A more recent study proposed that the structural change of the later element is triggered by a steric clash between the free catalytic lysine and Y322′ on the interfacial loop, which is induced by the conformational change of catalytic lysine in apo Cv-ωTA 24 . Moreover, this clash was also proposed to promote the dissociation of apo Cv-ωTA dimer.…”

Comparison of Four Immobilization Methods for Different Transaminases

“…The immobilization of enzymes via multipoint attachments is generally found to be more stable to external influences [75,85,86], which might improve the reusability of some enzymes. In addition, for transaminases, the dissociation of dimers upon the loss of the cofactor leaving inactive enzymes [7,87,88] might be reduced, leading to higher operational stability and thus, better reusability. On the other hand, if the enzymes are too densely packed, rigidified, and overcrowded, which is likely to occur in CLEAs and on beads, the total activity might decrease [85].…”

The Role of Buffer, Pyridoxal 5’‐Phosphate and Light on the Stability of the Silicibacter Pomeroyi Transaminase

“…In our case however, a precipitate was not visible after enzyme inactivation upon storage at room temperature. Here the inactivation was possibly caused by the dissociation of the enzymatic dimer which forms the active site [27] since enzyme activity is only possible in the dimeric form. The dissociation will lead to an opening of the “phosphate group binding cup”, which separates the residues important for the catalytic process and the PLP retention, rendering the enzyme inactive [22,28,29] .…”