Cloning, Baeyer-Villiger biooxidations, and structures of the camphor pathway 2-Oxo-\u3943-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A monooxygenase of Pseudomonas putida ATCC 17453

摘要

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-\u3943-4,5,5-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-\u3943-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli, and determined its three-dimensional structure with bound FAD at 1.95 \uc5 resolution as well as with bound FAD and NADP+ at 2.0 \uc5 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-\u3943-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 \ud7 105 M\u22121s\u22121) as a substrate, although its affinity (Km = 32 \u3bcM) was lower than that of the CoA-activated substrate (18 \u3bcM). 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.