Abstract
Methanol dehydrogenase (MDH), an NAD+-dependent oxidoreductase, reversibly converts formaldehyde to methanol. This activity is a key step for both toxic formaldehyde elimination and methanol production in bacterial methylotrophy. We mutated decameric Bacillus methanolicus MDH by directed evolution and screened mutants for increased formaldehyde reduction activity in Escherichia coli. The mutant with the highest formaldehyde reduction activity had three amino acid substitutions: F213V, F289L, and F356S. To identify the individual contributions of these residues to the increased reduction activity, the activities of mutant variants were evaluated. F213V/F289L and F213V/F289L/F356S showed 25.3- and 52.8-fold higher catalytic efficiency (kcat/Km) than wild type MDH, respectively. In addition, they converted 5.9- and 6.4-fold more formaldehyde to methanol in vitro than the wild type enzyme. Computational modelling revealed that the three substituted residues were located at MDH oligomerization interfaces, and may influence oligomerization stability: F213V aids in dimer formation, and F289L and F356S in decamer formation. The substitutions may stabilise oligomerization, thereby increasing the formaldehyde reduction activity of MDH.
Highlights
Increased atmospheric CO2 is a major cause of global warming and climate change
When we treated E. coli BL21 (DE3) cells expressing wild type Methanol dehydrogenase (MDH) with various concentration of formaldehyde, cell growth was arrested at concentrations >2 mM
The polymerase chain reaction (PCR) products were cloned into the pET21b plasmid under the control of a T7 promoter, and approximately 3 × 104 BL21 (DE3) transformants were grown in media containing 3 mM formaldehyde
Summary
Screening of MDH mutants with increased formaldehyde reduction activity. To develop a screening system for MDH mutants with increased formaldehyde reduction activity, we exploited the cytotoxicity differences between formaldehyde and methanol in Escherichia coli. To analyse which amino acid changes contributed to increased formaldehyde reduction activity, the three mutations were introduced separately and in combination to wild type MDH to form F213V, F289L, F356S, F213V/F289L, F289L/F356S, F213V/F356S, and F213V/F289L/F356S variants. The mutant containing all three amino acid substitutions exhibited the highest catalytic efficiency, with a 52.8-fold increase compared with wild type MDH. To examine the roles of the amino acid residues changed in MDH mutants, protein structure analysis was performed. Among mutants with only one amino acid change, only F213V showed higher catalytic activity than wild type MDH (Table 1). F289 and F356 were both located in the valley between dimers, and may influence decamer formation
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