Developing a biological reducing system for use at elevated temperatures

Abstract

S‐Adenosylmethionine (AdoMet)‐dependent radical enzymes constitute a rapidly expanding family of enzymes, the Radical SAM superfamily, that are involved in a variety of biological pathways. A defining characteristic of AdoMet‐dependent radical enzymes is the ability to use a reduced [4Fe‐4S] cluster to reductively cleave AdoMet, producing methionine and a 5′‐ deoxyadenosyl radical. This highly oxidizing radical intermediate initiates chemistry by abstracting a hydrogen atom from the substrate. Currently, the most common experimental means of reducing the [4Fe‐4S] cluster are the use of chemical reductants such as sodium dithionite, or the biochemical reductant flavodoxin. In the E. coli reducing system, NADPH reduces ferredoxin(flavodoxin) oxidoreductase (FNR), which then reduces flavodoxin (Fld) or ferredoxin (Fd). The E. coli system has drawbacks including thermal instability, and poor reaction kinetics. The development of a reducing system involving enzymes from thermophilic photosynthetic cyanobacteria will offer solutions to these problems, as such enzymes are stable and functional at elevated temperatures and exhibit rapid turnover due to their involvement in photosynthetic electron transport. We report that FNR from the thermophilic cyanobacterium, Thermosynechococcus elongatus, can be reduced by NADPH and rapidly transfers electrons to E. coli flavodoxin as well as T. elongatus ferredoxin. The component proteins are stable and active up to at least 65 °C. A heterologous reducing system comprised of NADPH, T. elongatus FNR, and E. coli flavodoxin is able to support the catalytic activity of biotin synthase from E. coli, as well as from thermophilic bacteria, including Methylococcus capsulatus. (Supported by NSF Grant MCB 09–23829 to J.T.J.)