A Versatile Disulfide-Driven Recycling System for NADP+ with High Cofactor Turnover Number

Antonio Angelastro, William M. Dawson, Louis Y.P. Luk, Rudolf K. Allemann*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

18 Citations (Scopus)
146 Downloads (Pure)

Abstract

NADP+-dependent enzymes are important in many biocatalytic processes to generate high-value chemicals for the pharmaceutical and food industry; hence, a cost-effective, efficient, and environmentally friendly recycling system for the relatively expensive and only marginally stable enzyme cofactor NADP+ offers significant benefits. NADP+ regeneration schemes have previously been described, but their application is severely limited by the low total turnover numbers (TTN) for the cofactor. Here, we report a glutathione-based recycling system that combines glutaredoxin from E. coli (EcGRX) and the glutathione reductase from S. cerevisiae (ScGR) for NADP+ regeneration. This system employs inexpensive latent organic disulfides such as oxidized cysteine or 2-hydroxyethyl disulfide (HED) as oxidizing agents and allows NADP+ recycling under both aerobic and anaerobic conditions with a TTN in excess of 5 × 105, indicating that each regeneration cycle is 99.9998% selective toward forming the cofactor. Accordingly, for each 1 mol of product generated, less than $0.05 of cofactor is needed. Finally, the EcGRX/ScGR pair is compatible with eight enzymes in the guanosine monophosphate (GMP) biosynthetic pathway, giving the corresponding isotopically labeled nucleotide in high yield. The glutathione-based NADP+ recycling system has potential for biocatalytic applications in academic and industrial settings.

Original languageEnglish
Pages (from-to)1025-1029
Number of pages5
JournalACS Catalysis
Volume7
Issue number2
Early online date19 Dec 2016
DOIs
Publication statusPublished - 3 Feb 2017

Keywords

  • biocatalysis
  • biosynthesis
  • biotechnology
  • cofactor/coenzyme recycling
  • enzyme oxidation

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