Abstract
Bioconversion of C1 chemicals such as methane and methanol into higher carbon-chain chemicals has been widely studied. Methanol oxidation catalyzed by methanol dehydrogenase (Mdh) is one of the key steps in methanol utilization in bacterial methylotrophy. In bacteria, few NAD+-dependent Mdhs have been reported that convert methanol to formaldehyde. In this study, an uncharacterized Mdh gene from Lysinibacillus xylanilyticus (Lxmdh) was cloned and expressed in Escherichia coli. The maximum alcohol oxidation activity of the recombinant enzyme was observed at pH 9.5 and 55°C in the presence of 10 mM Mg2+. To improve oxidation activity, rational approach-based, site-directed mutagenesis of 16 residues in the putative active site and NAD+-binding region was performed. The mutations S101V, T141S, and A164F improved the enzyme’s specific activity toward methanol compared to that of the wild-type enzyme. These mutants show a slightly higher turnover rate than that of wild-type, although their KM values were increased compared to that of wild-type. Consequently, according the kinetic results, S101, T141, and A164 positions may related to the catalytic activity in the active site for methanol dehydrogenation. It should be further studied other mutant variants with high activity for methanol. In conclusion, we characterized a new Lxmdh and its variants that may be potentially useful for the development of synthetic methylotrophy in the future.
Highlights
Methanol dehydrogenase (Mdh) catalyzes the interconversion of methanol and formaldehyde via an oxidation-reduction reaction
Sequence alignment of Lysinibacillus xylanilyticus derived methanol dehydrogenase (Lxmdh) revealed that 59% (222/378), 42% (160/381), 62% (240/385), and 40% (154/381) amino acid sequence identity with methanol dehydrogenase (Mdh) from B. methanolicus, B. stearothermophilus, C. necator and to lactaldehyde reductase (FucO) from E. coli, respectively (Figure 1)
To confirm the size of quaternary structure of Lxmdh, soluble protein was purified from the crude extract obtained from harvested cells by immobilized metal affinity chromatography (IMAC) purification system
Summary
Methanol dehydrogenase (Mdh) catalyzes the interconversion of methanol and formaldehyde via an oxidation-reduction reaction. Methanol is oxidized by Mdh; efficient methanol oxidation and concomitant formaldehyde assimilation and dissimilation is critical for the growth and energy generation of these organisms. In this regard, Mdh is a crucial enzyme for Abbreviations: ACT, endogenous activator protein; IMAC, immobilized metal affinity chromatography; Lxmdh, Lysinibacillus xylanilyticus derived methanol dehydrogenase; Mdh, methanol dehydrogenase; PQQ, pyrroloquinoline quinone. Alcohol oxidase in the peroxisome of yeast can oxidize alcohols including methanol to aldehyde and hydrogen peroxide, which are highly toxic to cells These two types of Mdhs require oxygen and move to specific cellular locations for their proper function. NAD+-dependent Mdh may be the best candidate for synthetic methylotrophy because it can perform its function under both aerobic and anaerobic conditions and generate reducing equivalents (NADH), which can help promote strain growth (Zhang et al, 2017)
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