In vitro incorporation of nascent molybdenum cofactor into human sulfite oxidase.

Published

Journal Article

We were able to reconstitute molybdopterin (MPT)-free sulfite oxidase in vitro with the molybdenum cofactor (Moco) synthesized de novo from precursor Z and molybdate. MPT-free human sulfite oxidase apoprotein was obtained by heterologous expression in an Escherichia coli mutant with a defect in the early steps of MPT biosynthesis. In vitro reconstitution of the purified apoprotein was achieved using an incubation mixture containing purified precursor Z, purified MPT synthase, and sodium molybdate. In vitro synthesized MPT generated from precursor Z by MPT synthase remains bound to the synthase. Surprisingly, MPT synthase was found capable of donating bound MPT to MPT-free sulfite oxidase. MPT was not released from MPT synthase when either bovine serum albumin or Moco-containing sulfite oxidase was used in place of aposulfite oxidase. After the inclusion of sodium molybdate in the reconstitution mixture, active sulfite oxidase was obtained, revealing that in vitro MPT synthase and aposulfite oxidase are sufficient for the insertion of MPT into sulfite oxidase and the conversion of MPT into Moco in the presence of high concentrations of molybdate. The conversion of MPT into Moco by molybdate chelation apparently occurs concomitantly with the insertion of MPT into sulfite oxidase.

Full Text

Duke Authors

Cited Authors

  • Leimkühler, S; Rajagopalan, KV

Published Date

  • January 19, 2001

Published In

Volume / Issue

  • 276 / 3

Start / End Page

  • 1837 - 1844

PubMed ID

  • 11042213

Pubmed Central ID

  • 11042213

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M007304200

Language

  • eng

Conference Location

  • United States