Engineering Oxygen-Independent NADH Oxidase Integrated with Electrocatalytic FAD Cofactor Regeneration

Mengjie Hou, Jing Yuan, Xinyu Dong, Yingjie Wang, Shihe Yang, Jiali Gao

Research output: Contribution to journalArticlepeer-review

Abstract

An electrochemically mediated enzyme process for nicotinamide adenine dinucleotide (NADH) oxidation and biosensing has been developed in which the oxygen-dependent activities of wild-type NADH oxidase are replaced by electrochemical regeneration of the flavin adenine dinucleotide (FAD) cofactor in the active site. Consequently, the present bioelectrocatalysis does not rely on a continuous oxygen supply through bubbling air or pure oxygen in biosynthetic applications, which reduces enzyme stability. The coupled electrochemical and enzymatic catalysis is achieved through a combination of enzyme immobilization on the electrode and electrochemical oxidation of FADH2 in the active site mediated by the electron transfer mediator ferrocene carboxylic acid (FcCA). Furthermore, to minimize the effect of dissolved oxygen when the electrocatalytic process is exposed to air, we successfully designed mutations at the Leu40 and Cys42 sites of Leuconostoc mesenteroides (LmNOx) to block the oxygen passage into the active site and to eliminate the native FAD cofactor regeneration half-reaction. The engineered enzymes, whose activities are significantly reduced or inactive in solution, are electrocatalytically active toward conversion of NADH to NAD+, demonstrating successful FAD cofactor regeneration in the active site via electrochemistry. Finally, we developed two highly responsive electrochemical biosensors for NADH detection which has a superior substrate specific to standard detectors using metal electrodes, and comparable detection range and detection limit (1-3 μM).

Original languageEnglish (US)
Pages (from-to)3581-3592
Number of pages12
JournalJACS Au
Volume4
Issue number9
DOIs
StatePublished - Sep 23 2024
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2024 The Authors. Published by American Chemical Society.

Keywords

  • NAD regeneration
  • electrochemical biosensors
  • mediated electron transfer (MET)
  • protein design
  • water-forming NADH oxidase (NOx)

PubMed: MeSH publication types

  • Journal Article

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