Polycyclic Ketone Monooxygenase from the Thermophilic Fungus Thermothelomyces thermophila: A Structurally Distinct Biocatalyst for Bulky Substrates

Abstract

Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a well-established tool to address these challenges, but known BVMOs have shortcomings in either stability or substrate selectivity. We characterized a novel BVMO from the thermophilic fungus Thermothelomyces thermophila, determined its three-dimensional structure, and demonstrated its use as a promising biocatalyst. This fungal enzyme displays excellent enantioselectivity, acts on various ketones, and is particularly active on polycyclic molecules. Most notably we observed that the enzyme can perform oxidations on both the A and D ring when converting steroids. These functional properties can be linked to unique structural features, which identify enzymes acting on bulky substrates as a distinct subgroup of the BVMO class.

Application of a thermostable Baeyer–Villiger monooxygenase for the synthesis of branched polyester precursors

“…insertion of one atom of oxygen at the most‐ or least‐substituted position, respectively). BVMOs display a broad substrate scope,4, 5 which include cyclic ketones with a heteroatom,6, 7 macrocyclic ketones,8, 9, 10, 11 and substituted derivatives of various ring size (for example, terpene‐based substrates12, 13). 14, 15 BVMOs are also especially interesting in the synthesis of branched lactones, from which branched aliphatic polyesters with low glass transition temperatures can be made 16, 17, 18, 19, 20, 21, 22, 23, 24, 25…”

Natural Variation in the ‘Control Loop’ of BVMOAFL210 and Its Influence on Regioselectivity and Sulfoxidation

“…The lack of structural data, and poor alignments due to variation in the sequence length of the 'control loop' in these BVMOs, does however leave uncertainty as to the correlations between sequence and structure. Structural studies with polycyclic ketone monooxygenase (PockeMO) revealed a Tyr to occupy the equivalent position in structure space [20]. In an attempt to elucidate the role of Thr513 in the 'control loop' of BVMO AFL210 , we therefore turned to determining the structure using X-ray crystallography.…”

Exploring the Substrate Scope of Baeyer–Villiger Monooxygenases with Branched Lactones as Entry towards Polyesters

“…Enzymes : AcCHMO, RhCHMO, TmCHMO, and PsCPDMO were prepared as purified His‐PTDH‐fusion proteins as previously described . E. coli NEB 10 beta cells transformed with the pCRE‐BVMO plasmid were grown for 40 h at 24 °C shaking in Terrific Broth (TB) medium supplemented with ampicillin and 0.02 % l ‐arabinose.…”

“…The abnormal products have been reported, for example, on rac ‐bicyclo[3.2.0]heptanone and on terpenone precursors, and stand in contrast to chemical Baeyer–Villiger oxidations, which typically yield either the normal lactone (i.e., the lactone with substituents next to the ester group) or a mixture of both lactones . In the past decades, numerous BVMOs have been discovered and described, thereby broadening the substrate scope of this family of enzymes with macrocyclic ketones, bi‐ and tricyclic compounds, steroids, heteroatom‐containing ketones, and substituted cyclic ketones derivatives …”