DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615) and XP_003665713 (MtVAO713), were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid.

L-Amino acid oxidase (LAAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme active on most proteinogenic L-amino acids, catalysing their conversion to α-keto acids by oxidative deamination of the substrate. For this oxidation reaction, molecular oxygen is used as the electron acceptor, generating hydrogen peroxide. LAAO can be used to detect L-amino acids, for the production of hydrogen peroxide as an oxidative agent or antimicrobial agent, and for the production of enantiopure amino acids from racemates. In this work, we characterised a previously reported LAAO from the bacterium Pseudoalteromonas luteoviolacea. The substrate scope and kinetic properties of the enzyme were determined, and the thermostability was evaluated. Additionally, we elucidated the crystal structure of this bacterial LAAO, enabling us to test the role of active site residues concerning their function in catalysis. The obtained insights and ease of expression of this thermostable LAAO provides a solid basis for the development of engineered LAAO variants tuned for biosensing and/or biocatalysis.

The creation of enzymes containing non-biological functionalities with activation modes outside of Nature’s canon paves the way towards fully programmable biocatalysis. Here, we present a fully genetically encoded boronic acid containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts. This boron enzyme catalyzes the kinetic resolution of hydroxyketones by oxime formation where crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign lead to a variant with natural enzyme like enantioselectivities for a number of different substrates. The unique activation mode of the boron enzyme was studied via X-ray crystallography, high resolution mass spectrometry and 11B NMR spectroscopy and opens up the possibility for a new class of boron dependent biocatalysts.

The evolution of a promiscuous enzyme for its various activities often results in catalytically specialized variants. This is an important natural mechanism to ensure the proper functioning of natural metabolic networks. It also acts as both a curse and blessing for enzyme engineers, where enzymes that have undergone directed evolution may exhibit exquisite selectivity at the expense of a diminished overall catalytic repertoire. We previously performed two independent directed evolution campaigns on a promiscuous artificial enzyme that leverages the unique properties of a non-canonical amino acid (ncAA) para- aminophenylalanine (pAF) as catalytic residue, resulting in two evolved variants which are both catalytically specialized. Here, we combine mutagenesis, crystallography and computation to reveal the molecular basis of the specialization phenomenon. In one evolved variant, an unexpected change in quaternary structure biases substrate dynamics to promote enantioselective catalysis, whilst the other demonstrates synergistic cooperation between natural side chains and the pAF residue to form semi-synthetic catalytic machinery. Our analysis provides valuable insights for the future engineering of effective artificial enzymes which employ either the widely used LmrR scaffold or pAF catalytic residue.

The catalytic repertoire of nature has been expanded over the past decades by the introduction of artificial metalloenzymes. These are enzymes containing a synthetic metal complex or a non-native metal ion. However, combining noble metal catalysis and enzymes remains challenging due to the lack of suitable ligands to bind these complexes. So far, noble metal artificial metalloenzyme design mostly involves in vitro approaches of ligand anchoring, like covalent modification of a cysteine residue or via supramolecular assembly. Here, we show a facile strategy to anchor a variety of 4d and 5d-transition metal complexes via genetic incorporation of a thiophenolic metal-binding ligand. We created a methodology to efficiently incorporate 4-mercaptophenylalanine in a protein scaffold using the stop codon suppression technology. The incorporated non-canonical amino acid was capable of binding a variety of noble metal complexes. To showcase the catalytic applications of this methodology, we developed an artificial hydroaminase by binding gold ions to the thiophenol-containing protein. The benefit of in vivo incorporation of the ligand is demonstrated by the susceptibility of catalytic activity to the microenvironment around the metal site, which can be modulated by changing the position of the ligand within the protein or by mutation of residues in its proximity.