Complete pyridine-nucleotide-specific conversion of an NADH-dependent ferredoxin reductase

Akito Nishizawa, Ayaka Harada, Miki Senda, Yuka Tachihara, Daisuke Muramatsu, Shinya Kishigami, Shigemasa Mori, Keisuke Sugiyama, Toshiya Senda, Shigenobu Kimura

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3 Citations (Scopus)

Abstract

The coenzyme specificity of enzymes is one of the critical parameters for the engineered production of biological compounds using bacteria. SinceNADPH is produced abundantly in photosynthetic organisms, conversion of an NADH-specific enzyme into an NADPH-specific one is a useful approach for the efficient carbon-neutral production of biological compounds in photosynthetic organisms. In the present study, anNADH-specific ferredoxin reductase component, BphA4 of biphenyl dioxygenase BphA from Acidovorax sp. Strain KKS102, was changed to an NADPH-dependent form using a method combining structure-based systematic mutations and site-directed random mutagenesis. The resultant CRG mutant, in which Glu175-Thr176-Gln177 of an NADH-recognition loop in the wild-type BphA4 was replaced with Cys 175-Arg176-Gly177, was highly specific and active for NADPH, and its biochemical and structural properties for NADPH were nearly the same as those of the wild-type BphA4 for NADH. In addition, this mutation project was assessed by a semiempirical prediction method of mutation effects, and the results suggested that the CRG mutant was one of the best NADPHspecific mutants.

Original languageEnglish
Pages (from-to)257-265
Number of pages9
JournalBiochemical Journal
Volume462
Issue number2
DOIs
Publication statusPublished - 2014 Sep 1
Externally publishedYes

Keywords

  • Biphenyl degradation
  • Electron transfer
  • Flavoprotein
  • Protein design
  • Random mutagenesis
  • Specificity prediction

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Nishizawa, A., Harada, A., Senda, M., Tachihara, Y., Muramatsu, D., Kishigami, S., Mori, S., Sugiyama, K., Senda, T., & Kimura, S. (2014). Complete pyridine-nucleotide-specific conversion of an NADH-dependent ferredoxin reductase. Biochemical Journal, 462(2), 257-265. https://doi.org/10.1042/BJ20140384