Open AccessMolecular Cloning and Sequence Determination of the lpd Gene Encoding Lipoamide Dehydrogenase from Pseudomonas fluorescens
Author(s) -
J.A.E. Benen,
W.J.H. van Berkel,
W.M.A.M. van Dongen,
Florian Müller,
A. de Kok
Publication year - 1989
Publication title -
microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.019
H-Index - 179
eISSN - 1465-2080
pISSN - 1350-0872
DOI - 10.1099/00221287-135-7-1787
Subject(s) - dihydrolipoamide dehydrogenase , biology , microbiology and biotechnology , pseudomonas fluorescens , nucleic acid sequence , azotobacter vinelandii , dehydrogenase , gene , biochemistry , plasmid , genetics , enzyme , bacteria , nitrogenase , nitrogen fixation
The lpd gene encoding lipoamide dehydrogenase (dihydrolipoamide dehydrogenase; EC 1.8.1.4) was isolated from a library of Pseudomonas fluorescens DNA cloned in Escherichia coli TG2 by use of serum raised against lipoamide dehydrogenase from Azotobacter vinelandii. Large amounts (up to 15% of total cellular protein) of the P. fluorescens lipoamide dehydrogenase were produced by the E. coli clone harbouring plasmid pCJB94 with the lipoamide dehydrogenase gene. The enzyme was purified to homogeneity by a three-step procedure. The gene was subcloned from plasmid pCJB94 and the complete nucleotide sequence of the subcloned fragment (3610 bp) was determined. The derived amino acid sequence of P. fluorescens lipoamide dehydrogenase showed 84% and 42% homology when compared to the amino acid sequences of lipoamide dehydrogenase from A. vinelandii and E. coli, respectively. The lpd gene of P. fluorescens is clustered in the genome with genes for the other components of the 2-oxoglutarate dehydrogenase complex.