Overproduction, Purification, and Characterization of Chlorocatechol Dioxygenase, a Non-Heme Iron Dioxygenase with Broad Substrate Tolerance

Joan B. Broderick*, Thomas V O'Halloran

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

69 Scopus citations

Abstract

We show here that purified chlorocatechol dioxygenase from Pseudomonas putida is able to oxygenate a wide range of substituted catechols with turnover numbers ranging from 2 to 29 s−1. This enzyme efficiently cleaves substituted catechols bearing electron-donating or multiple electron-withdrawing groups in an intradiol manner with kcat/KM values between 0.2 × 107 and 1.4 × 107 M−1 s−1. These unique catalytic properties prompted a comparison with the related but highly specific enzymes catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase. The chlorocatechol dioxygenase gene (clcA) from the Pseudomonas plasmid pAC27 was subcloned into the expression vector pKK223-3, allowing production of chlorocatechol dioxygenase to approximately 7–8% of total cellular protein. An average of 4 mg of purified enzyme has been obtained per gram of wet cells. Protein and iron analyses indicate an iron stoichiometry of 1 iron/ 57.5-kDa homodimer, α2Fe. The electronic absorption spectrum contains a broad tyrosinate to iron charge transfer transition centered at 430 nm (є = 3095 M−1 cm−1 based on iron concentration) which shifts to 490 nm (є = 3380 M−1 cm-−1) upon catechol binding. The resonance Raman spectrum of the native enzyme exhibits characteristic tyrosine ring vibrations. Electron paramagnetic resonance data for the resting enzyme (g = 4.25, 9.83) is consistent with high-spin iron (III) in a rhombic environment. This similarity between the spectroscopic properties of the Fe(III) centers in chlorocatechol dioxygenase and the more specific dioxygenases suggests a highly conserved catalytic site. We infer that the unique catalytic properties of chlorocatechol dioxygenase are due to other characteristics of its substrate binding pocket.

Original languageEnglish (US)
Pages (from-to)7349-7358
Number of pages10
JournalBiochemistry
Volume30
Issue number29
DOIs
StatePublished - Jul 1 1991

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Overproduction, Purification, and Characterization of Chlorocatechol Dioxygenase, a Non-Heme Iron Dioxygenase with Broad Substrate Tolerance'. Together they form a unique fingerprint.

Cite this