High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor.

Published

Journal Article

The flavoprotein and hemoprotein components of Escherichia coli B NADPH-sulfite reductase are encoded by cysJ and cysI, respectively. Plasmids containing these two genes overexpressed flavoprotein catalytic activity and apohemoprotein by 13- to 35-fold, but NADPH-sulfite reductase holoenzyme activity was increased only 3-fold. Maximum overexpression of holoenzyme activity was achieved by the inclusion in such plasmids of Salmonella typhimurium cysG, which encodes a uroporphyrinogen III methyltransferase required for the synthesis of siroheme, a cofactor for the hemoprotein. Thus, cofactor deficiency, in this case siroheme, can limit overexpression of a cloned enzyme. Catalytically active holoenzyme accounted for 10% of total soluble protein in a host containing cloned cysJ, cysI, and cysG. A 5.3-kb DNA fragment containing S. typhimurium cysG was sequenced, and the open reading frame corresponding to cysG was identified by subcloning and by identifying plasmid-encoded peptides in maxicells. Comparison with the sequence reported for the E. coli cysG region (J. A. Cole, unpublished data; GenBank sequence ECONIRBC) indicates a gene order of nirB-nirC-cysG in the cloned S. typhimurium fragment. In addition, two open reading frames of unknown identity were found immediately downstream of cysG. One of these contains 11 direct repeats of 33 nucleotides each, which correspond to the consensus amino acid sequence Asp-Asp-Val-Thr-Pro-Pro-Asp-Asp-Ser-Gly-Asp.

Full Text

Duke Authors

Cited Authors

  • Wu, JY; Siegel, LM; Kredich, NM

Published Date

  • January 1, 1991

Published In

Volume / Issue

  • 173 / 1

Start / End Page

  • 325 - 333

PubMed ID

  • 1987123

Pubmed Central ID

  • 1987123

Electronic International Standard Serial Number (EISSN)

  • 1098-5530

International Standard Serial Number (ISSN)

  • 0021-9193

Digital Object Identifier (DOI)

  • 10.1128/jb.173.1.325-333.1991

Language

  • eng