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

Abstract

Baeyer-Villiger monooxygenases (BVMOs) are flavin-dependent enzymes that primarily convert ketones to esters, but can also catalyze heteroatom oxidation. Several structural studies have highlighted the importance of the 'control loop' in BVMOs, which adopts different conformations during catalysis. Central to the 'control loop' is a conserved tryptophan that has been implicated in NADP(H) binding. BVMO AFL210 from Aspergillus flavus, however, contains a threonine in the equivalent position. Here, we report the structure of BVMO AFL210 in complex with NADP + in both the 'open' and 'closed' conformations. In neither conformation does Thr513 contact the NADP + . Although mutagenesis of Thr513 did not significantly alter the substrate scope, changes in peroxyflavin stability and reaction rates were observed. Mutation of this position also brought about changes in the regioand enantioselectivity of the enzyme. Moreover, lower rates of overoxidation during sulfoxidation of thioanisole were also observed.

Enzymatic Kinetic Resolution by Addition of Oxygen

“…[33] BVMOs are versatile enzymes whose catalytic activity can be employed to synthesise valuable heteroatom-bearing chiral building blocks by non-BV processes.F or example, they are known to catalyse the oxidation of aw ide range of heteroatoms including nitrogen, selenium, boron, phosphorus,a nd sulfur. [34] Codexis used this reactivity in their largescale synthesis of the chiral sulfoxide Esomeprazole,using an engineered BVMO to circumvent problems associated with the previously used Kagan-Sharpless-Pitchen type oxida-…”

Editorial: Special Issue on “Flavin Monooxygenases”

“…Staying with the industrial application theme of BVMOs, Tolmie et al [6] discuss the natural variation in the "control loop" of another BVMO, BVMO AF210 from Aspergillus flavons and its influence on regioselectivity and sulfoxidation. The authors note that with the exception of directed evolution studies influencing the substrate scope and selectivity/specificity through allosteric effects, protein engineering of BVMOs to improve substrate acceptance or regio-, enantio-, and stereospecificity/selectivity has mostly focused on "hot spot" residues lining the active site.…”