Abstract
Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme.
Highlights
Amine oxidations are observed in a wide range of biological processes, including the metabolism of polyamines and neurotransmitters in higher organisms and as a source of ammonium in lower eukaryotes and bacteria
Overall Structure An examination of the three dimensional atomic structure of Cyclohexylamine oxidase (CHAO) reveals that the overall fold is substantially similar to that of the human monoamine oxidases (MAOs) B monomer, with a RMSD of 1.1 Afor the coordinates of the a-carbon atoms
Like monoamine oxidase B (MAO B), the CHAO structure is composed of two domains - a substratebinding region and a cofactor-binding domain, with the isoalloxazine moiety of flavin adenine dinucleotide (FAD) at their interface
Summary
Amine oxidations are observed in a wide range of biological processes, including the metabolism of polyamines and neurotransmitters in higher organisms and as a source of ammonium in lower eukaryotes and bacteria. The flavin-containing amine oxidases carry out the oxidative cleavage of the a-CH bond of the amine to form an imine product while reducing a flavin cofactor. This unstable compound undergoes aqueous hydrolysis to yield the corresponding aldehyde and either ammonia or an amine, depending on the substrate. The monoamine oxidases (MAOs) are flavin-containing amine oxidases that are widely distributed in higher eukaryotes where they oxidize the primary amino groups of arylalkyl amine substrates [4]. We present here a structural analysis of a bacterial cyclohexylamine oxidase (CHAO) in both the holoenzyme form with bound flavin adenine dinucleotide (FAD), as well as a ternary complex with FAD and the oxidation product cyclohexanone. We use random acceleration molecular dynamics (RAMD) to identify possible routes of exit for products from the active site and propose an egress mechanism based on these results
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