A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity.
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Toogood HS, Fryszkowska A, Hulley M, Sakuma M, Mansell D, Stephens GM, Gardiner JM, Scrutton NS
A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity.
Chembiochem. 2011 Mar 21;12(5):738-49. doi: 10.1002/cbic.201000662. Epub 2011 Mar 4.
- PubMed ID
- 21374779 [ View in PubMed]
- Abstract
We have conducted a site-specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of alpha,beta-unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2-phenyl-1-nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to CC reduction. These mutants showed an enhancement in a minor side reaction and formed 2-phenylpropanal oxime from 2-phenyl-1-nitropropene. The multiple binding conformations of hydroxy substituted nitro-olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.