title: Complete pyridine-nucleotide-specific conversion of an NADH-dependent ferredoxin reductase.
abstract: The coenzyme specificity of enzymes is one of the critical parameters for the engineered production of biological compounds using bacteria. Since NADPH 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, an NADH-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 Cys175-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 semi-empirical prediction method of mutation effects, and the results suggested that the CRG mutant was one of the best NADPH-specific mutants.
authors.affiliation: *VB, Organization for Creation of Collaboration and Innovation, Ibaraki University, Hitachi, Ibaraki 316-8511, Japan.
authors.forename: Akito
authors.initials: A
authors.lastname: Nishizawa
authors.forename: Ayaka
authors.initials: A
authors.lastname: Harada
authors.affiliation: †Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Senda
authors.affiliation: §Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki 316-8511, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Tachihara
authors.affiliation: §Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki 316-8511, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Muramatsu
authors.affiliation: ∥Graduate School of Life Sciences, University of Hyogo, 3-2-1 Kouto, Kamigori, Hyogo 678-1297, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Kishigami
authors.affiliation: §Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki 316-8511, Japan.
authors.forename: Shigemasa
authors.initials: S
authors.lastname: Mori
authors.affiliation: §Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki 316-8511, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Sugiyama
authors.forename: Toshiya
authors.initials: T
authors.lastname: Senda
authors.affiliation: §Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki 316-8511, Japan.
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Kimura
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D042621:Comamonadaceae / Q000201:enzymology*; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000737:chemistry*; D009249:NADP / Q000737:chemistry*; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009243:NAD; D009249:NADP; D049308:Dioxygenases; D005287:Ferredoxin-NADP Reductase