Structural and Catalytic Characterization of a Fungal Baeyer-Villiger Monooxygenase

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.

Aerobic Baeyer–Villiger Oxidation Catalyzed by a Flavin‐Containing Enzyme Mimic in Water

“…Example of natural BVMO bearing af lavin cofactor in its active site. [28] Before preparing the artificial flavoenzyme,P EI was randomly modified by the covalent incorporation of guanidinium and octyl groups and purified by dialysis as previously described. [24] After lyophilization from water,t he so-formed polymer was characterized by 1 HNMR spectroscopy,w hich allowed the determination of the guanidinium/octyl group ratio (see Figure S1 in the Supporting Information).…”

Alkyl Formate Ester Synthesis by a Fungal Baeyer–Villiger Monooxygenase

“…[14][15][16] Conversion of the aromatic aldehydes benzaldehyde and 4-hydroxybenzaldehyde results in carbon migration to form the corresponding ester, with autohydrolysis to the phenol product. [21,22] In order to investigate the substrate specificity and selectivity of BVMO AFL838 towards aldehydes,t he enzyme was purified to homogeneity,a nd biotransformations were performed with Bacillus megaterium glucose dehydrogenase (BmGDH)f or cofactor regeneration. Phenylacetaldehyde is converted with low selectivity for the acid product by cyclohexanonem onooxygenase (CHMO) and BVMO4 from Dietzia sp.…”

“…[16,18,19] It was recently demonstrated through mutational studies on CHMO that regioselectivity is mediated by substrate positioning in the active site due to stericc onstraints. [21,22] In order to investigate the substrate specificity and selectivity of BVMO AFL838 towards aldehydes,t he enzyme was purified to homogeneity,a nd biotransformations were performed with Bacillus megaterium glucose dehydrogenase (BmGDH)f or cofactor regeneration. [14,15] We therefore decidedt ot est our library of recombinant fungal BVMOs from Aspergillus flavus expressed in Escherichia coli ( Figure S2 in the Supporting Information) against aliphatic aldehydes with octanal as the test substrate ( Figure S3).…”

“…[22] Glucose dehydrogenase from B. megaterium (pET28:BmGDH) was similarly expressed and purified to homogeneity by Ni-affinity chromatography and desalted on aP D10 column (GE Healthcare). [22] Glucose dehydrogenase from B. megaterium (pET28:BmGDH) was similarly expressed and purified to homogeneity by Ni-affinity chromatography and desalted on aP D10 column (GE Healthcare).…”

“…BVMO AFL838 has previously been shown to catalyze the conversion of various2 -alkanones with absolute regioselectivity to the corresponding alkyl acetate products. [21,22] In order to investigate the substrate specificity and selectivity of BVMO AFL838 towards aldehydes,t he enzyme was purified to homogeneity,a nd biotransformations were performed with Bacillus megaterium glucose dehydrogenase (BmGDH)f or cofactor regeneration. Gratifyingly,a ll the aldehydes tested (Scheme 1) yielded the corresponding formate esters (Table 1).Complete or near-complete conversions of valeraldehyde (1a)a nd hexanal (2a)a nd more than 80 %c onversion of octanal (3a)w ere obtainedw ithin 2h.T hese products were slowly hydrolyzed to primary alcohols, with the shorter chain-length alkyl formatesg iving more of the hydrolyzed alcohol products.…”