3P67

T26S mutant of pentaerythritol tetranitrate reductase containing a bound acetate molecule

Summary for 3P67

• Entry DOI: 10.2210/pdb3p67/pdb
• Related: 1H50 3P62
• Descriptor: Pentaerythritol tetranitrate reductase, FLAVIN MONONUCLEOTIDE, ACETATE ION, ... (4 entities in total)
• Functional Keywords: old yellow enzyme family, alpha, beta barrel, oxidoreductase
• Biological source: Enterobacter cloacae
• Total number of polymer chains: 1
• Total formula weight: 41141.72

Authors

Toogood, H.S.,Scrutton, N.S. (deposition date: 2010-10-11, release date: 2010-11-24, Last modification date: 2023-11-01)

Primary citation

Hulley, M.E.,Toogood, H.S.,Fryszkowska, A.,Mansell, D.,Stephens, G.M.,Gardiner, J.M.,Scrutton, N.S.
Focused Directed Evolution of Pentaerythritol Tetranitrate Reductase by Using Automated Anaerobic Kinetic Screening of Site-Saturated Libraries
Chembiochem, 11:2433-2447, 2010

PubMed Abstract: This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96-well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site-saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)-2-aryl-1-nitropropene and α-methyl-trans-cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,β-unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.

PubMed: 21064170
DOI: 10.1002/cbic.201000527

Experimental method

X-RAY DIFFRACTION (1.5 Å)