6TB1

Crystal structure of thermostable omega transaminase 6-fold mutant from Pseudomonas jessenii

Summary for 6TB1

• Entry DOI: 10.2210/pdb6tb1/pdb
• Descriptor: Aspartate aminotransferase family protein, PYRIDOXAL-5'-PHOSPHATE, GLYCEROL, ... (6 entities in total)
• Functional Keywords: transaminase, aminotransferase, transferase
• Biological source: Pseudomonas sp.
• Total number of polymer chains: 2
• Total formula weight: 100884.67

Authors

Capra, N.,Rozeboom, H.J.,Thunnissen, A.M.W.H.,Janssen, D.B. (deposition date: 2019-10-31, release date: 2020-07-15, Last modification date: 2024-01-24)

Primary citation

Meng, Q.,Capra, N.,Palacio, C.M.,Lanfranchi, E.,Otzen, M.,van Schie, L.Z.,Rozeboom, H.J.,Thunnissen, A.W.H.,Wijma, H.J.,Janssen, D.B.
Robust omega-Transaminases by Computational Stabilization of the Subunit Interface.
Acs Catalysis, 10:2915-2928, 2020

PubMed Abstract: Transaminases are attractive catalysts for the production of enantiopure amines. However, the poor stability of these enzymes often limits their application in biocatalysis. Here, we used a framework for enzyme stability engineering by computational library design (FRESCO) to stabilize the homodimeric PLP fold type I ω-transaminase from . A large number of surface-located point mutations and mutations predicted to stabilize the subunit interface were examined. Experimental screening revealed that 10 surface mutations out of 172 tested were indeed stabilizing (6% success), whereas testing 34 interface mutations gave 19 hits (56% success). Both the extent of stabilization and the spatial distribution of stabilizing mutations showed that the subunit interface was critical for stability. After mutations were combined, 2 very stable variants with 4 and 6 mutations were obtained, which in comparison to wild type ( = 62 °C) displayed values of 80 and 85 °C, respectively. These two variants were also 5-fold more active at their optimum temperatures and tolerated high concentrations of isopropylamine and cosolvents. This allowed conversion of 100 mM acetophenone to ()-1-phenylethylamine (>99% enantiomeric excess) with high yield (92%, in comparison to 24% with the wild-type transaminase). Crystal structures mostly confirmed the expected structural changes and revealed that the most stabilizing mutation, I154V, featured a rarely described stabilization mechanism: namely, removal of steric strain. The results show that computational interface redesign can be a rapid and powerful strategy for transaminase stabilization.

PubMed: 32953233
DOI: 10.1021/acscatal.9b05223

Experimental method

X-RAY DIFFRACTION (1.85 Å)