Heterologous expression of alkene monooxygenase components from Xanthobacter autotrophicus Py2 and reconstitution of the active complex

Verawat Champreda, Ning Yi Zhou, David J. Leak

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The coupling protein and ferredoxin from Xanthobacter autotrophicus Py2 alkene monooxygenase (Xamo) have been functionally expressed in both N-terminal affinity tagged fusion and native forms in Escherichia coli. However, attempts to express the NADH-oxidoreductase and oxygenase, always resulted in the production of inactive, insoluble proteins. Nevertheless, the recombinant reductase from the toluene 4-monooxygenase of Pseudomonas mendocina KR1 was found to functionally complement the Xamo system. In vitro reconstitution, using the recombinant coupling protein and other components purified from the wild type, showed that steady-state epoxidation rate and coupling efficiency were dependent on the relative concentration of Xamo components in the reaction. The optimal molar stoichiometric ratio of Xamo components was determined to be approximately 1:0.25-0.3:2:2 (oxygenase hexamer:reductase:ferredoxin:coupling protein), suggesting the formation of an efficient catalytic complex at the minimal stoichiometric ratio to saturate the probable two-fold symmetry binding sites on the oxygenase.

Original languageEnglish
Pages (from-to)309-318
Number of pages10
JournalFEMS Microbiology Letters
Volume239
Issue number2
DOIs
Publication statusPublished - 1 Oct 2004

Fingerprint

Xanthobacter
Oxygenases
Ferredoxins
Oxidoreductases
Pseudomonas mendocina
Proteins
Recombinant Proteins
NAD
Binding Sites
Escherichia coli
alkene monooxygenase

Keywords

  • Component interaction
  • Coupling protein
  • Ferredoxin
  • Monooxygenase
  • Xanthobacter

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Applied Microbiology and Biotechnology
  • Microbiology

Cite this

Heterologous expression of alkene monooxygenase components from Xanthobacter autotrophicus Py2 and reconstitution of the active complex. / Champreda, Verawat; Zhou, Ning Yi; Leak, David J.

In: FEMS Microbiology Letters, Vol. 239, No. 2, 01.10.2004, p. 309-318.

Research output: Contribution to journalArticle

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