Helix Zipper Regulating Formolase Activity

Abstract

Formolase catalyzes the carboligation of C1 compound formaldehyde, providing a unique opportunity for producing multicarbon compounds with formaldehyde as the sole substrate. Elucidating the molecular rationale determining the activity of formolase, especially at low concentrations of formaldehyde, is essential for improving its catalytic performance in the C1 compound conversion. In this study, a channel-modulating helix formed a zipper structure with its neighboring helix that was identified to regulate the catalytic activity of formolase_BFD, especially at low concentrations of formaldehyde. Up to 24.1-fold increased catalytic efficiency was obtained after engineering the channel-modulating helix to fine-tune the shape of substrate/product channel. The activity of the best variant was enhanced by 27.3-fold at 20 mM formaldehyde and 86.5-fold at 40 mM formaldehyde compared to the starting point. Crystallization and molecular dynamic simulations revealed that the engineered zipper structure enhanced the activity of formolase_BFD by enlarging the substrate/product channel entrance and tightening the bottom of the channel, which increased the availability of substrate formaldehyde to promote the formation of C3 product 1,3-dihydroxyacetone.