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Modulating proposed electron transfer pathways in P450BM3 led to improved activity and coupling efficiency.

Authors
  • Darimont, Dominique1
  • Weissenborn, Martin J2
  • Nebel, Bernd A1
  • Hauer, Bernhard3
  • 1 Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. , (Germany)
  • 2 Leibniz-Institute of Plant Biochemistry (IPB), Weinberg 3, 06120 Halle (Saale), Germany; Martin-Luther-University Halle-Wittenberg, Institute of Chemistry, Germany. , (Germany)
  • 3 Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Electronic address: [email protected] , (Germany)
Type
Published Article
Journal
Bioelectrochemistry (Amsterdam, Netherlands)
Publication Date
Feb 01, 2018
Volume
119
Pages
119–123
Identifiers
DOI: 10.1016/j.bioelechem.2017.08.009
PMID: 28965071
Source
Medline
Keywords
License
Unknown

Abstract

Electrochemical in vitro reduction of P450 enzymes is a promising alternative to in vivo applications. Previously we presented three engineered P450BM3 variants for aniline hydroxylation, equipped with a carbon nanotube binding-peptide (CNT-tag) for self-assembly on CNT electrodes. Compared to wildtype P450BM3 the NADPH-dependent activity was enhanced, but the coupling efficiency remained low. For P450BM3 Verma, Schwaneberg and Roccatano (2014, Biopolymers 101, 197-209) calculated putative electron transfer pathways (eTPs) by MD simulations. We hypothesised that knockouts of these transfer pathways would alter the coupling efficiency of the system. The results revealed no improved system for the electrically-driven P450s. For the NADPH-driven P450s, however, the most active eTP-mutant showed a 13-fold increased activity and a 32-fold elevated coupling efficiency using NADPH as reducing equivalent. This suggests an alternative principle of electron transport for the reduction by NADPH and an electrode, respectively. The work presents moreover a tool to improve the coupling and activity of P450s with non-natural substrates.

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