Abstract
Catalytic promiscuity is a promising starting point for improving the existing enzymes and even creating novel enzymes. In this work, site-directed mutagenesis was performed to improve promiscuous alcohol dehydrogenase activity of reductive aminase from Aspergillus oryzae (AspRedAm). AspRedAm showed the cofactor preference toward NADPH in reductive aminations, while it favored NADH in the reduction reactions. Some key amino acid residues such as N93, I118, M119, and D169 were identified for mutagenesis by molecular docking. Variant N93A showed the optimal pH and temperature of 8 and 30°C, respectively, in the reduction of 5-hydroxymethylfurfural (HMF). The thermostability was enhanced upon mutation of N93 to alanine. The catalytic efficiency of variant N93A (k cat/K m, 23.6 mM−1 s−1) was approximately 2-fold higher compared to that of the wild-type (WT) enzyme (13.1 mM−1 s−1). The improved catalytic efficiency of this variant may be attributed to the reduced steric hindrance that stems from the smaller side chain of alanine in the substrate-binding pocket. Both the WT enzyme and variant N93A had broad substrate specificity. Escherichia coli (E. coli) cells harboring plain vector enabled selective reduction of biobased furans to target alcohols, with the conversions of 35–95% and the selectivities of >93%. The introduction of variant N93A to E. coli resulted in improved substrate conversions (>98%) and selectivities (>99%).
Highlights
Enzymes are well known for their exquisite chemo, regio, and stereoselectivities
We recently found that a Reductive AminaseReductive aminases (RedAms) from Aspergillus oryzae (AspRedAm) (Aleku et al, 2017) possessed promiscuous alcohol dehydrogenase (ADH) activity, which enabled 5hydroxymethylfurfural (HMF) to be selectively but slowly reduced to 2,5-bis(hydroxymethyl)furan (BHMF)
Based on the crystal structure of AspRedAm (Aleku et al, 2017) and the results of this protein docking with HMF and cofactor (Supplementary Figure 1), the residues within 4 Å around substrate HMF such as N93, I118, M119, A120, V121, and D169 were identified as the potential hotspots for mutation, since they may interact with substrate and cofactor
Summary
Enzymes are well known for their exquisite chemo-, regio-, and stereoselectivities. In addition to the reactions that they have evolved for, more enzymes have been found to be capable of promiscuously catalyzing mechanistically distinct transformations (termed catalytic promiscuity) (Kazlauskas, 2005). Reductive aminases (RedAms), a subclass of imine reductases (IREDs), have evolved to be capable of catalytic formation of imines as well as their reduction, namely, reductive amination reactions (Aleku et al, 2017; Sharma et al, 2017; Ducrot et al, 2020). Lenz et al described the generation of new IREDs from β-hydroxyacid dehydrogenases by single amino acid substitutions (Lenz et al, 2018). Amine dehydrogenases (AmDHs), a type of useful enzymes catalyzing the conversion of ketones to enantiomerically pure amines, were created from amino acid dehydrogenases (Tseliou et al, 2019; Liu et al, 2020). Tseliou reported that L-lysineε-dehydrogenase variants showing dual AmDH/ADH activities were applied for direct conversion of alcohols to amines using a single enzyme (Tseliou et al, 2020)
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