Abstract
Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that convert ketones to esters. Due to their high regio-, stereo- and enantioselectivity and ability to catalyse these reactions under mild conditions, they have gained interest as alternatives to chemical Baeyer-Villiger catalysts. Despite their widespread occurrence within the fungal kingdom, most of the currently characterized BVMOs are from bacterial origin. Here we report the catalytic and structural characterization of BVMOAFL838 from Aspergillus flavus. BVMOAFL838 converts linear and aryl ketones with high regioselectivity. Steady-state kinetics revealed BVMOAFL838 to show significant substrate inhibition with phenylacetone, which was more pronounced at low pH, enzyme and buffer concentrations. Para substitutions on the phenyl group significantly improved substrate affinity and increased turnover frequencies. Steady-state kinetics revealed BVMOAFL838 to preferentially oxidize aliphatic ketones and aryl ketones when the phenyl group are separated by at least two carbons from the carbonyl group. The X-ray crystal structure, the first of a fungal BVMO, was determined at 1.9 Å and revealed the typical overall fold seen in type I bacterial BVMOs. The active site Arg and Asp are conserved, with the Arg found in the “in” position. Similar to phenylacetone monooxygenase (PAMO), a two residue insert relative to cyclohexanone monooxygenase (CHMO) forms a bulge within the active site. Approximately half of the “variable” loop is folded into a short α-helix and covers part of the active site entry channel in the non-NADPH bound structure. This study adds to the current efforts to rationalize the substrate scope of BVMOs through comparative catalytic and structural investigation of different BVMOs.
Highlights
Baeyer-Villiger monooxygenases (BVMOs) are flavin-dependent enzymes that catalyze the conversion of ketones to esters using NAD(P)H and molecular oxygen [1,2,3,4]
BVMOAFL838 was heterologously expressed in E. coli as a C-terminally 6x histidine tagged protein and purified to near homogeneity using Ni-affinity and size exclusion chromatography (SEC)
BVMOAFL838 was recovered with only 70–80% of bound flavin, but near full occupancy of the FAD could be achieved by soaking with excess FAD before SEC (Fig 1B)
Summary
Baeyer-Villiger monooxygenases (BVMOs) are flavin-dependent enzymes that catalyze the conversion of ketones to esters using NAD(P)H and molecular oxygen [1,2,3,4]. In addition to this typical reaction, they can catalyze heteroatom oxidation, including sulfoxidation and N-oxidation, as well as epoxidation reactions. The substrate scope of the collective BVMO family of enzymes has grown to include a variety of substrates ranging from acetone to larger ketones such as steroids. Many directed evolution studies have been performed to increase or alter the substrate scope as well as improve the selectivity and specificity of these enzymes [5,6].
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