Journal
CHEMBIOCHEM
Volume 12, Issue 5, Pages 738-749Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201000662
Keywords
asymmetric bioreduction; biocatalysis; PETN reductase; unsaturated alkenes; X-ray crystallography
Funding
- UK Biotechnology and Biological Sciences Research Council (BBSRC)
- Biotechnology and Biological Sciences Research Council [BB/G023581/2, BB/D002826/1, BB/G023581/1, BB/E010717/1, BB/D01963X/1] Funding Source: researchfish
- BBSRC [BB/G023581/2, BB/D01963X/1, BB/D002826/1, BB/G023581/1, BB/E010717/1] Funding Source: UKRI
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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 C=C 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.
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