Properties of a proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli with alpha and beta subunits linked through fused transmembrane helices

• Proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of an alpha and a beta subunit, whereas the homologues mitochondrial enzyme contains a single polypeptide. As compared to the latter transhydrogenase, using a 14-helix model for its membrane topology, the point of fusion is between the transmembrane helices 4 and 6 where the fusion linker provides the extra transmembrane helix 5. In order to clarify the potential role of this extra helix/linker, the alpha and the beta subunits were fused using three connecting peptides of different lengths, one (pAX9) involving essentially a direct coupling, a second (pKM) with a linking peptide of 18 residues, and a third (pKMII) with a linking peptide of 32 residues, as compared to the mitochondrial extra peptide of 27 residues. The results demonstrate that the plasma membrane-bound and purified pAX9 enzyme with the short linker was partly misfolded and strongly inhibited with regard to both catalytic activities and proton translocation, whereas the properties of pKM and pKMII with longer linkers were similar to those of wild-type E coli transhydrogenase but partly different from those of the mitochondrial enzyme although pKMII generally gave higher activities. It is concluded that a mitochondrial-like linking peptide is required for proper folding and activity of the E coli fused transhydrogenase, and that differences between the catalytic properties of the E coli and the mitochondrial enzymes are unrelated to the linking peptide. This is the first time that larger subunits of a membrane protein with multiple transmembrane helices have been fused with retained activity. (C) 2001 Published by Elsevier Science B.V.

Subject headings

NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

Keyword

transhydrogenase

nicotinamide adenine dinucleotide

nicotinamide

adenine dinucleotide

reduced

proton pump

membrane protein

SITE-DIRECTED MUTAGENESIS

PURIFICATION

MITOCHONDRIA

ACID

NAD

Publication and Content Type

ref (subject category)

art (subject category)