3UP4

Crystal Structure of OTEMO complex with FAD and NADP (form 3)

Summary for 3UP4

• Entry DOI: 10.2210/pdb3up4/pdb
• Related: 3UOV 3UOX 3UOY 3UOZ 3UP5
• Descriptor: OTEMO, FLAVIN-ADENINE DINUCLEOTIDE, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, ... (4 entities in total)
• Functional Keywords: baeyer-villiger monooxygenase, oxidoreductase
• Biological source: Pseudomonas putida
• Total number of polymer chains: 2
• Total formula weight: 125949.81

Authors

Shi, R.,Matte, A.,Cygler, M.,Lau, P. (deposition date: 2011-11-17, release date: 2012-02-01, Last modification date: 2024-10-30)

Primary citation

Leisch, H.,Shi, R.,Grosse, S.,Morley, K.,Bergeron, H.,Cygler, M.,Iwaki, H.,Hasegawa, Y.,Lau, P.C.
Cloning, Baeyer-Villiger biooxidations, and structures of the camphor pathway 2-oxo-{Delta}(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase of Pseudomonas putida ATCC 17453.
Appl.Environ.Microbiol., 78:2200-2212, 2012

PubMed Abstract: A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140-152, 1983). Here we cloned and overexpressed the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP(+) at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP(+). A comparison of several crystal forms of OTEMO bound to FAD and NADP(+) revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (k(cat)/K(m)) favors 2-n-hexyl cyclopentanone (4.3 × 10(5) M(-1) s(-1)) as a substrate, although its affinity (K(m) = 32 μM) was lower than that of the CoA-activated substrate (K(m) = 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 extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

PubMed: 22267661
DOI: 10.1128/AEM.07694-11

Experimental method

X-RAY DIFFRACTION (2.804 Å)