6tb0

Crystal structure of thermostable omega transaminase 4-fold mutant from Pseudomonas jessenii

spinqualitylabelsall models 6tb0, resolution 1.95Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6tb0 is a 2 chain structure with sequence from Pseudomonas sp.. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.95Å Ligands: GOL, PLP, SIN Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed 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 omega-transaminase from Pseudomonas jessenii. 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 (T m (app) = 62 degrees C) displayed T m (app) values of 80 and 85 degrees 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 (S)-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. Robust omega-Transaminases by Computational Stabilization of the Subunit Interface.,Meng Q, Capra N, Palacio CM, Lanfranchi E, Otzen M, van Schie LZ, Rozeboom HJ, Thunnissen AWH, Wijma HJ, Janssen DB ACS Catal. 2020 Mar 6;10(5):2915-2928. doi: 10.1021/acscatal.9b05223. Epub 2020, Jan 31. PMID:32953233 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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