Completing the series of BVMOs involved in camphor metabolism of Pseudomonas putida NCIMB 10007 by identification of the two missing genes, their functional expression in E. coli, and biochemical characterization

Appl Microbiol Biotechnol. 2012 Oct;96(2):419-29. doi: 10.1007/s00253-011-3859-1.

Abstract

The camphor-degrading Baeyer-Villiger monooxygenases (BVMOs) from Pseudomonas putida NCIMB 10007 have been of interest for over 40 years. So far the FMN- and NADH-dependent type II BVMO 3,6-diketocamphane 1,6-monooxygenase (3,6-DKCMO) and the FAD- and NADPH-dependent type I BVMO 2-oxo-∆3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) have not been entirely studied, since it was not possible to produce those enzymes in satisfactory amounts and purity. In this study, we were able to clone and recombinantly express both enzymes and subsequently use them as biocatalysts for various mono- and bicyclic ketones. Full conversion could be reached with both enzymes towards (±)-cis-bicyclo[3.2.0]hept-2-en-6-one and with 3,6-DKCMO towards (−)-camphor. Further OTEMO gave full conversion with norcamphor. OTEMO was found to have a pH optimum of 9 and a temperature optimum of 20 °C and converted (±)-cis-bicyclo[3.2.0]hept-2-en-6-one with a k cat/K M value of 49.3 mM-1 s-1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Camphor / chemistry
  • Camphor / metabolism*
  • Enzyme Stability
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Gene Expression
  • Kinetics
  • Mixed Function Oxygenases / chemistry*
  • Mixed Function Oxygenases / genetics*
  • Mixed Function Oxygenases / metabolism
  • Molecular Sequence Data
  • Phylogeny
  • Pseudomonas putida / chemistry
  • Pseudomonas putida / classification
  • Pseudomonas putida / enzymology*
  • Pseudomonas putida / genetics
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Camphor
  • Mixed Function Oxygenases

Associated data

  • GENBANK/JQ034404
  • GENBANK/JQ034405