Cloning, Baeyer-Villiger biooxidations, and structures of the camphor pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A monooxygenase of Pseudomonas putida ATCC 17453

Download
  1. Get@NRC: Cloning, Baeyer-Villiger biooxidations, and structures of the camphor pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A monooxygenase of Pseudomonas putida ATCC 17453 (Opens in a new window)
DOIResolve DOI: http://doi.org/10.1128/AEM.07694-11
AuthorSearch for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for:
TypeArticle
Journal titleApplied and Environmental Microbiology
Volume78
Issue7
Pages22002212; # of pages: 13
AbstractA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA, a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453 had been initially characterized in 1983 by Trudgill and co-workers (H.J. Ougham, D.G. Taylor, and P.W. Trudgill, J. Bacteriol. 153:140-152, 1983). Here we have cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli, and determined its three-dimensional structure with bound FAD at 1.95 Å resolution as well as with bound FAD and NADP+ at 2.0 Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. Comparison of several crystal forms of OTEMO bound to FAD and NADP+ revealed conformational plasticity of several loop regions, some of which have been implicated as contributing to the substrate specificity profile of structurally-related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105 M−1s−1) as a substrate, although its affinity (Km = 32 μM) was lower than that of the CoA-activated substrate (18 μM). In whole cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO), and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work expands our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs as well as expanding the catalytic repertoire of one of its original members.
Publication date
LanguageEnglish
AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedYes
NRC number53169
NPARC number20220776
Export citationExport as RIS
Report a correctionReport a correction
Record identifier8e280ea6-d150-4e2b-a1c7-a2cf5b8cc571
Record created2012-06-30
Record modified2016-05-09
Bookmark and share
  • Share this page with Facebook (Opens in a new window)
  • Share this page with Twitter (Opens in a new window)
  • Share this page with Google+ (Opens in a new window)
  • Share this page with Delicious (Opens in a new window)