Construction and characterization of novel bifunctional fusion proteins composed of alcohol dehydrogenase and NADH oxidase with efficient oxidized cofactor regeneration

Abstract

To tune the efficiency of oxidized cofactor recycling between alcohol dehydrogenase (ADH) and NADH oxidase (NOX) for the production of aromatic chiral alcohols, we designed and constructed four novel bifunctional fusion proteins composed of thermostable ADH and NOX from Thermococcus kodakarensis KOD1. ADH was linked to the N-or C-terminus of NOX with a typical rigid linker (EAAAK) 3 and a flexible linker (GGGGS) 3 , respectively. Compared with the parental enzymes, the NOX moieties in the four fusion proteins exhibited higher specific activities (141%-282%), while the ADH moieties exhibited varying levels of specific activity (69%-167%). All fusion proteins showed decreased affinities toward the cofactors, with increased K m values toward NADH (159%-406%) and NAD + (202%-372%). In the enantioselective oxidation of (RS)-1-phenylethanol coupled with cofactor regeneration, the four fusion proteins displayed different positive and negative effects on the recycling efficiency of the oxidized cofactor. The two fusion proteins composed of NOX at the N-terminus exhibited higher total turnover numbers than the corresponding mixtures of individual enzymes with equal activities, particularly at low cofactor concentrations. These findings suggest high cofactor recycling efficiencies of the fusion proteins with appropriate design and their potential application in the biosynthesis of chiral alcohols.

Engineered repeat proteins as scaffolds to assemble multi-enzyme systems for efficient cell-free biosynthesis

“…4a). Through UV-vis quantification of the produced resorufin, we calculated the H 2 O 2 generated and consequently, the NADH consumed, by NOX 36,37 . Hence, the more active the NOX is, the less efficient the NADH channeling is between FDH1 and AlaDH3.…”

“…A competition test 36 was designed to assess a potential substrate channeling effect 60 . Pure NADH oxidase (NOX) from Thermus thermophilus 61 was added as a competitor to the multi-enzymatic reaction catalyzed by the scaffolded FDH1 AlaDH3 enzymes.…”

Microbial alcohol dehydrogenases: recent developments and applications in asymmetric synthesis

“…Interestingly, two fusion proteins, NOX-R3-ADH and NOX-F3-ADH, with NOX at the N-terminus, catalysed the enantioselective oxidation of (S)-82 with higher cofactor recycling efficiencies than those of the corresponding individual enzyme mixtures (Scheme 20). 136 In addition to commonly known cofactor regenerating enzymes like GDH, LDH and FDH, some special dehydrogenases also have been identified and utilized in enzyme-coupled cofactor regeneration approaches. For example, Jia et al recently engineered an isopropanol dehydrogenase from Brucella suis (IPADH) to regenerate NAD(P) cofactors efficiently.…”

“…Interestingly, two fusion proteins, NOX-R3-ADH and NOX-F3-ADH, with NOX at the N-terminus, catalysed the enantioselective oxidation of ( S )- 82 with higher cofactor recycling efficiencies than those of the corresponding individual enzyme mixtures (Scheme 20). 136…”