A Highly Active and Specific Benzoylformate Decarboxylase for Glycolaldehyde Production from Formaldehyde
Junhui Zhou, Florian Bourdeaux, Vincent R. Emann, Tianwei Tan, Ulrich Schwaneberg
Abstract
Coupling C1 fixation to the central metabolism, for example, via the synthetic acetyl-CoA (SACA) pathway requires enzymes that catalyze the condensation of two formaldehyde (FALD) molecules to one glycolaldehyde (GALD) molecule without the formation of any side products such as 1,3-dihydroxyacetone and erythrulose. The benzoylformate decarboxylase from Polynucleobacter necessarius (PnBFD) is a promising candidate to produce GALD from FALD without side products, but its use is limited by its pathetic specific activity (5.91 U/g). Through a three-step protein engineering campaign combined with a systematic study on the role of the noncatalytic cofactor, Mg 2+, the PnBFD-M1 (V355I/G398W/S431A/E469A) variant was generated. PnBFD-M1 showed a 247-fold improved initial activity (1118.56 U/g) and a high product specificity (99.5% GALD), the highest reported to date for BFDs. Analysis of the structure and MD simulations identified three potential factors for boosting the PnBFD-M1 activity: increased hydrophobicity, reduced flexibility of the active pocket and the substrate channel, and reduced distance of the catalytic cofactor (thiamine diphosphate) to the catalytic amino acids caused by the exchange from Mg 2+ to Fe 2+ .