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In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen
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Spatial requirement for PAMO for transformation of non-native linear substrates
Alexandra T P Carvalho1, Daniel F A R Dourado, Timofey Skvortsov
1School of Chemistry and Chemical Engineering, Queen's University, David Keir Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, UK. m.huang@qub.ac.uk.
Physical Chemistry Chemical Physics : PCCP
|January 11, 2018
Summary
Phenylacetone monooxygenase (PAMO) engineering enhances its ability to convert linear substrates. A key mutation repositions L289, enabling stable substrate binding for improved industrial biocatalysis and potential biodiesel production.
Area of Science:
- Biocatalysis
- Enzyme Engineering
- Molecular Dynamics
Background:
- Phenylacetone monooxygenase (PAMO) is a stable Baeyer-Villiger monooxygenase ideal for ester and lactone synthesis.
- Limited substrate scope hinders PAMO's industrial application, particularly for linear substrates.
Purpose of the Study:
- To identify spatial requirements for PAMO to convert non-native linear substrates.
- To understand the mechanism behind enhanced activity in a quadruple PAMO variant.
Main Methods:
- Molecular dynamics simulations comparing WT PAMO and a quadruple variant (P253F/G254A/R258M/L443F) with 2-octanone and phenylacetone.
- Steady-state kinetic analysis of single-mutation variants.
Main Results:
- A significant repositioning of L289 in the quadruple variant reshapes the active site, crucial for binding aliphatic substrates like 2-octanone.
- This L289 movement prevents substrate dissociation from the catalytic site, enabling efficient oxygenation.
- Kinetic analysis confirmed that quadruple mutations collectively drive the L289 reposition.
Conclusions:
- Atomic-level insights into PAMO's active site dynamics facilitate rational enzyme engineering.
- This study paves the way for expanding PAMO's substrate scope for industrial biocatalysis, including potential biodiesel applications.
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