Norcoclaurine synthase (NCS) catalyzes a stereoselective Pictet-Spengler reaction to give the key intermediate, (S)-norcoclaurine in benzylisoquinoline alkaloid (BIA) biosynthesis. This family of alkaloids contains many bioactive molecules including morphine and berberine. Recently, NCS has been demonstrated to accept a variety of aldehydes and some

Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interact efficiently with our designed peptides and assemble the Escherichia coli and related orthologs clamps, whereas group II clamps poorly interact with the same peptides and include Bacillus subtilis and other Gram-positive clamps. These studies also suggest that the peptide binding process could occur via different mechanisms, which depend on the type of clamp.

Vanillyl alcohol oxidase (VAO) is a fungal flavoenzyme that converts a wide range of para-substituted phenols. The products of these conversions, e.g. vanillin, coniferyl alcohol and chiral aryl alcohols, are of interest for several industries. VAO is the only known fungal member of the 4-phenol oxidising (4PO) subgroup of the VAO/PCMH flavoprotein family. While the enzyme has been biochemically characterised in great detail, little is known about its physiological role and distribution in fungi. We have identified and analysed novel, fungal candidate VAOs and found them to be mostly present in Pezizomycotina and Agaricomycotina. The VAOs group into three clades, of which two clades do not have any characterised member. Interestingly, bacterial relatives of VAO do not form a single outgroup, but rather split up into two separate clades. We have analysed the distribution of candidate VAOs in fungi, as well as their genomic environment. VAOs are present in low frequency in species of varying degrees of relatedness and in regions of low synteny. These findings suggest that fungal VAOs may have originated from bacterial ancestors, obtained by fungi through horizontal gene transfer. Because the overall conservation of fungal VAOs varies between 60 and 30% sequence identity, we argue for a more reliable functional prediction using critical amino acid residues. We have defined a sequence motif P-x-x-x-x-S-x-G-[RK]-N-x-G-Y-G-[GS] that specifically recognizes 4PO enzymes of the VAO/PCMH family, as well as additional motifs that can help to further narrow down putative functions. We also provide an overview of fingerprint residues that are specific to VAOs.

Vanillyl alcohol oxidase (VAO) is a homo-octameric flavoenzyme belonging to the VAO/PCMH family. Each VAO subunit consists of two domains, the FAD-binding and the cap domain. VAO catalyses, among other reactions, the two-step conversion of p-creosol (2-methoxy-4-methylphenol) to vanillin (4-hydroxy-3-methoxybenzaldehyde). To elucidate how different ligands enter and exit the secluded active site, Monte Carlo based simulations have been performed. One entry/exit path via the subunit interface and two additional exit paths have been identified for phenolic ligands, all leading to the si side of FAD. We argue that the entry/exit path is the most probable route for these ligands. A fourth path leading to the re side of FAD has been found for the co-ligands dioxygen and hydrogen peroxide. Based on binding energies and on the behaviour of ligands in these four paths, we propose a sequence of events for ligand and co-ligand migration during catalysis. We have also identified two residues, His466 and Tyr503, which could act as concierges of the active site for phenolic ligands, as well as two other residues, Tyr51 and Tyr408, which could act as a gateway to the re side of FAD for dioxygen. Most of the residues in the four paths are also present in VAO’s closest relatives, eugenol oxidase and p-cresol methylhydroxylase. Key path residues show movements in our simulations that correspond well to conformations observed in crystal structures of these enzymes. Preservation of other path residues can be linked to the electron acceptor specificity and oligomerisation state of the three enzymes. This study is the first comprehensive overview of ligand and co-ligand migration in a member of the VAO/PCMH family, and provides a proof of concept for the use of an unbiased method to sample this process.

The VAO/PCMH family of flavoenzymes is a family of structurally related proteins that catalyse a wide range of oxidation reactions. It contains a subfamily of enzymes that catalyse the oxidation of para-substituted phenols using covalently bound FAD cofactors (the 4PO subfamily). This subfamily is composed of two oxidases, vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO), and two flavocytochrome dehydrogenases, paracresol methylhydroxylase (PCMH) and eugenol hydroxylase (EUGH). Although they catalyse similar reactions, these enzymes differ in terms of their electron acceptor preference and oligomerization state. For example, VAO forms homo-octamers that can be described as tetramers of stable dimers, whereas EUGO is exclusively dimeric in solution. A possible explanation for this difference is the presence of a loop at the dimer-dimer interface in VAO that is not present in EUGO. Here, the role played by this loop in determining the quaternary structure of these enzymes is investigated. A VAO variant where the loop was deleted, loopless VAO, exclusively formed dimers. However, introduction of the loop into EUGO was not sufficient to induce its octamerization. Neither variant displayed major changes in its catalytic properties as compared to the wild-type enzyme. Bioinformatic analysis revealed that the presence of the loop is conserved within putative fungal oxidases of the 4PO subgroup, but it is never found in putative bacterial oxidases or dehydrogenases. Our results shed light on the molecular mechanism of homo-oligomerization of VAO and the importance of oligomerization for its enzymatic function.Enzymes p-cresol methylhydroxylase (4-methylphenol:acceptor oxidoreductase (methyl-hydroxylating), EC 1.17.99.1); vanillyl alcohol oxidase (vanillyl alcohol:oxygen oxidoreductase, EC 1.1.3.38).