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Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1
Ist Teil von
Applied microbiology and biotechnology, 2016-06, Vol.100 (11), p.4969-4983
Ort / Verlag
Berlin/Heidelberg: Springer Berlin Heidelberg
Erscheinungsjahr
2016
Link zum Volltext
Quelle
SpringerLink (Online service)
Beschreibungen/Notizen
Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD
+
as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the
Bacillus methanolicus
Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from
Cupriavidus necator
N-1 (previously designated as
Ralstonia eutropha
). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as
B. methanolicus
ACT and
Escherichia coli
Nudix hydrolase NudF, or putative native
C. necator
activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the
B. methanolicus
Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher
K
cat
/
K
m
for methanol and 10-fold lower
K
cat
/
K
m
for
n
-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.