Genome mining leads to the identification of a stable and promiscuous Baeyer-Villiger monooxygenase from a thermophilic microorganism.

Baeyer-Villiger monooxygenases (BVMOs) are NAD(P)H-dependent flavoproteins that convert ketones to esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer-Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel BVMOs that are both stable and promiscuous. Our genome mining led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssnBVMO that exhibits i) the highest melting temperature of any naturally sourced BVMO (62.5 ºC), ii) a remarkable kinetic stability across a wide range of conditions, similar to those of PAMO and PockeMO, iii) optimal catalysis at 50 °C, and iv) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Subsequent quantitative assays using propiophenone demonstrated >95% conversion. Several fusions were also constructed that linked ssnBVMO to a thermostable phosphite dehydrogenase. These fusions can recycle NADPH and catalyze oxidations with sub-stoichiometric quantities of this expensive cofactor. Characterization of these fusions permitted identification of PTDH-L1-ssnBVMO as the most promising protein that could have utility as a seed sequence for enzyme engineering campaigns aiming to develop biocatalysts for Baeyer-Villiger oxidations.

A simple and direct ionic chromatography method to monitor galactose oxidase activity.

Galactose oxidase (GalOx, EC.1.1.3.9) is one of the most extensively studied copper radical oxidases (CROs). The reaction catalyzed by GalOx leads to the oxidation of the C-6 hydroxyl group of galactose and galactosides (including galactosylated polysaccharides and glycoproteins) to the corresponding aldehydes, coupled to the reduction of dioxygen to hydrogen peroxide. Despite more than 60 years of research including mechanistic studies, enzyme engineering and application development, GalOx activity remains primarily monitored by indirect measurement of the co-product hydrogen peroxide. Here, we describe a simple direct method to measure GalOx activity through the identification of galactosylated oxidized products using high-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD). Using galactose and lactose as representative substrates, we were able to separate and detect the C-6 oxidized products, which were confirmed by LC-MS and NMR analyses to exist in their hydrated (geminal-diol) forms. We show that the HPAEC-PAD method is superior to other methods in terms of sensitivity as we could detect down to 0.08 µM of LacOX (eq. 30 µg L-1). We believe the method will prove useful for qualitative detection of galactose oxidase activity in biological samples or for quantitative purposes to analyze enzyme kinetics or to compare enzyme variants in directed evolution programs.

Active-Site Engineering Switches Carbohydrate Regiospecificity in a Fungal Copper Radical Oxidase.

Copper radical oxidases (CROs) from Auxiliary Activity Family 5, Subfamily 2 (AA5_2), are organic cofactor-free biocatalysts for the selective oxidation of alcohols to the corresponding aldehydes. AA5_2 CROs comprise canonical galactose-6-oxidases as well as the more recently discovered general alcohol oxidases and aryl alcohol oxidases. Guided by primary and tertiary protein structural analyses, we targeted a distinct extended loop in the active site of a Colletotrichum graminicola aryl alcohol oxidase (CgrAAO) to explore its effect on catalysis in the broader context of AA5_2. Deletion of this loop, which is bracketed by a conserved disulfide bridge, significantly reduced the inherent activity of the enzyme toward extended galacto-oligosaccharides, as anticipated from molecular modeling. Unexpectedly, kinetic and product analysis on a range of monosaccharides and disaccharides revealed that an altered carbohydrate specificity in CgrAAO-Δloop was accompanied by a complete change in regiospecificity from C-6 to C-1 oxidation, thereby generating aldonic acids. C-1 regiospecificity is unprecedented in AA5 enzymes and is classically associated with flavin-dependent carbohydrate oxidases of Auxiliary Activity Family 3. Thus, this work further highlights the catalytic adaptability of the unique mononuclear copper radical active site and provides a basis for the design of improved biocatalysts for diverse potential applications.

A survey of substrate specificity among Auxiliary Activity Family 5 copper radical oxidases.

There is significant contemporary interest in the application of enzymes to replace or augment chemical reagents toward the development of more environmentally sound and sustainable processes. In particular, copper radical oxidases (CRO) from Auxiliary Activity Family 5 Subfamily 2 (AA5_2) are attractive, organic cofactor-free catalysts for the chemoselective oxidation of alcohols to the corresponding aldehydes. These enzymes were first defined by the archetypal galactose-6-oxidase (GalOx, EC 1.1.3.13) from the fungus Fusarium graminearum. The recent discovery of specific alcohol oxidases (EC 1.1.3.7) and aryl alcohol oxidases (EC 1.1.3.47) within AA5_2 has indicated a potentially broad substrate scope among fungal CROs. However, only relatively few AA5_2 members have been characterized to date. Guided by sequence similarity network and phylogenetic analysis, twelve AA5_2 homologs have been recombinantly produced and biochemically characterized in the present study. As defined by their predominant activities, these comprise four galactose 6-oxidases, two raffinose oxidases, four broad-specificity primary alcohol oxidases, and two non-carbohydrate alcohol oxidases. Of particular relevance to applications in biomass valorization, detailed product analysis revealed that two CROs produce the bioplastics monomer furan-2,5-dicarboxylic acid (FDCA) directly from 5-hydroxymethylfurfural (HMF). Furthermore, several CROs could desymmetrize glycerol (a by-product of the biodiesel industry) to D- or L-glyceraldehyde. This study furthers our understanding of CROs by doubling the number of characterized AA5_2 members, which may find future applications as biocatalysts in diverse processes.