Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase

Flavin-dependent carbohydrate oxidases are valuable tools in biotechnological applications due to their high selectivity in the oxidation of carbohydrates. In this study, we report the biochemical and structural characterization of a recently discovered carbohydrate oxidase from the bacterium Ralstonia solanacearum, which is a member of the vanillyl alcohol oxidase flavoprotein family. Due to its exceptionally high activity toward N-acetyl-d-galactosamine and N-acetyl-d-glucosamine, the enzyme was named N-acetyl-glucosamine oxidase (NagOx). In contrast to most known (fungal) carbohydrate oxidases, NagOx could be overexpressed in a bacterial host, which facilitated detailed biochemical and enzyme engineering studies. Steady state kinetic analyses revealed that non-acetylated hexoses were also accepted as substrates albeit with lower efficiency. Upon determination of the crystal structure, structural insights into NagOx were obtained. A large cavity containing a bicovalently bound FAD, tethered via histidyl and cysteinyl linkages, was observed. Substrate docking highlighted how a single residue (Leu251) plays a key role in the accommodation of N-acetylated sugars in the active site. Upon replacement of Leu251 (L251R mutant), an enzyme variant was generated with a drastically modified substrate acceptance profile, tuned toward non-N-acetylated monosaccharides and disaccharides. Furthermore, the activity toward bulkier substrates such as the trisaccharide maltotriose was introduced by this mutation. Due to its advantage of being overexpressed in a bacterial host, NagOx can be considered a promising alternative engineerable biocatalyst for selective oxidation of monosaccharides and oligosaccharides.</p

Discovery and structural characterization of a thermostable bacterial monoamine oxidase

Monoamine oxidases (MAOs) are pivotal regulators of neurotransmitters in mammals, while microbial MAOs have been shown to be valuable biocatalysts for enantioselective synthesis of pharmaceutical compounds or precursors thereof. To extend the knowledge of how MAOs function at the molecular level and in order to provide more biocatalytic tools, we set out to identify and study a robust bacterial variant: a MAO from the thermophile Thermoanaerobacterales bacterium (MAO(Tb)). MAO(Tb) is highly thermostable with melting temperatures above 73 degrees C and is well expressed in Escherichia coli. Substrate screening revealed that the oxidase is most efficient with n-alkylamines with n-heptylamine being the best substrate. Presteady-state kinetic analysis shows that reduced MAO(Tb) rapidly reacts with molecular oxygen, confirming that it is a bona fide oxidase. The crystal structure of MAO(Tb) was resolved at 1.5 & Aring; and showed an exceptionally high similarity with the two human MAOs, MAO A and MAO B. The active site of MAO(Tb) resembles mostly the architecture of human MAO A, including the cysteinyl protein-FAD linkage. Yet, the bacterial MAO lacks a C-terminal extension found in human MAOs, which explains why it is expressed and purified as a soluble protein, while the mammalian counterparts are anchored to the membrane through an alpha-helix. MAO(Tb) also displays a slightly different active site access tunnel, which may explain the specificity toward long aliphatic amines. Being an easy-to-express, thermostable enzyme, for which a high-resolution structure was elucidated, this bacterial MAO may develop into a valuable biocatalyst for synthetic chemistry or biosensing