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 Kcat/Km for methanol and 10-fold lower Kcat/Km 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.
Submitter: Marie Ary
Submission Date: June 26, 2017, 3:05 p.m.
|Number of data points||193|
|Proteins||NAD-dependent methanol dehydrogenase Mdh2 ; Zn-dependent alcohol dehydrogenase AdhA|
|Assays/Quantities/Protocols||Experimental Assay: Nash activity (relative to WT) ; Experimental Assay: kcat Methanol ; Experimental Assay: Vmax ; Experimental Assay: Butanol specific activity ; Experimental Assay: Ethanol specific activity ; Experimental Assay: Km Methanol ; Experimental Assay: Propanol specific activity ; Experimental Assay: kcat n-Butanol ; Experimental Assay: Km n-Butanol ; Experimental Assay: Methanol specific activity ; Derived Quantity: kcat/Km n-Butanol ; Derived Quantity: kcat/Km Methanol ; Derived Quantity: kcat/Km methanol fold change (relative to WT) ; Derived Quantity: MeOH/BuOH activity ratio ; Derived Quantity: MeOH/EtOH activity ratio ; Derived Quantity: MeOH/PrOH activity ratio|
|Libraries||Kinetic parameters of engineered Mdh2 variants to methanol and n-butanol, using NADH as cofactor (Table 4) ; Effect of A169 replacement on Mdh2 methanol specificity (Table 5) ; C1 to C4 alcohol specific activity and MeOH/C2-C4 alcohol activity ratios (Fig. 6) ; Relative Nash activity of all A169 pt variants (Fig. S2) ; Kinetic parameters for AdhA from C. glutamicum (Table 6)|