Cloning and Analysis of the Genes for a Novel Electron-transferring Flavoprotein from Megasphaera elsdenii: EXPRESSION AND CHARACTERIZATION OF THE RECOMBINANT PROTEIN

Hugh O'Neill,Stephen G Mayhew,Geraldine Butler

doi:10.1074/jbc.273.33.21015

Hugh O'Neill, Stephen G Mayhew + Show 1 more

Open Access

https://doi.org/10.1074/jbc.273.33.21015

Copy DOI

Abstract

The genes that encode the two different subunits of the novel electron-transferring flavoprotein (ETF) from Megasphaera elsdenii were identified by screening a partial genomic DNA library with a probe that was generated by amplification of genomic sequences using the polymerase chain reaction. The cloned genes are arranged in tandem with the coding sequence for the beta-subunit in the position 5' to the alpha-subunit coding sequence. Amino acid sequence analysis of the two subunits revealed that there are two possible dinucleotide-binding sites on the alpha-subunit and one on the beta-subunit. Comparison of M. elsdenii ETF amino acid sequence to other ETFs and ETF-like proteins indicates that while homology occurs with the mitochondrial ETF and bacterial ETFs, the greatest similarity is with the putative ETFs from clostridia and with fixAB gene products from nitrogen-fixing bacteria. The recombinant ETF was isolated from extracts of Escherichia coli. It is a heterodimer with subunits identical in size to the native protein. The isolated enzyme contains approximately 1 mol of FAD, but like the native protein it binds additional flavin to give a total of about 2 mol of FAD/dimer. It serves as an electron donor to butyryl-CoA dehydrogenase, and it also has NADH dehydrogenase activity.

Highlights

Electron-transferring flavoproteins (ETFs)1 catalyze electron transfer between other flavoproteins [1]
Comparison of M. elsdenii ETF amino acid sequence to other ETFs and ETF-like proteins indicates that while homology occurs with the mitochondrial ETF and bacterial ETFs, the greatest similarity is with the putative ETFs from clostridia and with fixAB gene products from nitrogen-fixing bacteria
A sequence similarity search of the protein/DNA data bases using the BLAST search algorithm [47] revealed that the N-terminal sequence of the ␤-subunit of M. elsdenii ETF is encoded by a region downstream from the gene encoding butyryl-CoA dehydrogenase [48]

Summary

Introduction

Electron-transferring flavoproteins (ETFs) catalyze electron transfer between other flavoproteins [1]. The pig liver enzyme is the best characterized ETF It is found in the mitochondrion, where it functions to transfer electrons from flavoprotein dehydrogenases involved in the metabolism of fatty acids, choline, and amino acids to a further flavoprotein, named ETF-CoQ oxidoreductase. The latter enzyme passes the electrons to the terminal electron transfer chain [1]. There is evidence that they are artifacts generated during isolation of this ETF [22] Their optical properties differ from those of FAD, and they affect the catalytic properties of the enzyme.

Methods

Results

Conclusion