Structural and Functional Characteristics of FMN-Dependent NADPH-Indigo Reductase Homolog from Bacillus cohnii

Abstract

We found indigo reductase homolog in Bacillus cohnii gene and succeeded in production of a large amount of the recombinant homolog in Escherichia coli. The homolog exhibited FMN-dependent NADPH-quinone reductase activity, but not indigo-reducing activity. Crystal structure analysis of the enzyme revealed the formation of a binary complex with FMN, 2-propanol, and glycerol, determined at a resolution of 1.57 Å. Notably, the structure of FMN was of particular interest, as the isoalloxazine ring of FMN exhibited a butterfly-like bent conformation, with an angular deviation of approximately 9.4º to 10.9º along the axis between N5 and N10. The reason for FMN adopting a butterfly-like structure was thought that the exposure of the enzyme crystal to X-ray radiation led to the one-electron reduction of FMN, forming the semiquinone radical FMNH^•.