Advances in the understanding and exploitation of carbohydrate-active enzymes.

Carbohydrate-active enzymes (CAZymes) are responsible for the biosynthesis, modification and degradation of all glycans in Nature. Advances in genomic and metagenomic methodologies, in conjunction with lower cost gene synthesis, have provided access to a steady stream of new CAZymes with both well-established and novel mechanisms. At the same time, increasing access to cryo-EM has resulted in exciting new structures, particularly of transmembrane glycosyltransferases of various sorts. This improved understanding has resulted in widespread progress in applications of CAZymes across diverse fields, including therapeutics, organ transplantation, foods, and biofuels. Herein, we highlight a few of the many important advances that have recently been made in the understanding and applications of CAZymes.

Characterization of a new family of 6-sulfo-N-acetylglucosaminidases.

Sulfation is widespread in nature and plays an important role in modulating biological function. Among the strategies developed by microbes to access sulfated oligosaccharides as a nutrient source is the production of 6-sulfoGlcNAcases to selectively release 6-sulfoGlcNAc from target oligosaccharides. Thus far, all 6-sulfoGlcNAcases identified have belonged to the large GH20 family of ß-hexosaminidases. Ηere, we identify and characterize a new, highly specific non-GH20 6-sulfoGlcNAcase from Streptococcus pneumoniae TIGR4, Sp_0475 with a greater than 110,000-fold preference toward N-acetyl-ß-D-glucosamine-6-sulfate substrates over the nonsulfated version. Sp_0475 shares distant sequence homology with enzymes of GH20 and with the newly formed GH163 family. However, the sequence similarity between them is sufficiently low that Sp_0475 has been assigned as the founding member of a new glycoside hydrolase family, GH185. By combining results from site-directed mutagenesis with mechanistic studies and bioinformatics we provide insight into the substrate specificity, mechanism, and key active site residues of Sp_0475. Enzymes of the GH185 family follow a substrate-assisted mechanism, consistent with their distant homology to the GH20 family, but the catalytic residues involved are quite different. Taken together, our results highlight in more detail how microbes can degrade sulfated oligosaccharides for nutrients.

Carbohydrate-active enzymes (CAZymes) in the gut microbiome.

The 1013-1014 microorganisms present in the human gut (collectively known as the human gut microbiota) dedicate substantial percentages of their genomes to the degradation and uptake of carbohydrates, indicating the importance of this class of molecules. Carbohydrates function not only as a carbon source for these bacteria but also as a means of attachment to the host, and a barrier to infection of the host. In this Review, we focus on the diversity of carbohydrate-active enzymes (CAZymes), how gut microorganisms use them for carbohydrate degradation, the different chemical mechanisms of these CAZymes and the roles that these microorganisms and their CAZymes have in human health and disease. We also highlight examples of how enzymes from this treasure trove have been used in manipulation of the microbiota for improved health and treatment of disease, in remodelling the glycans on biopharmaceuticals and in the potential production of universal O-type donor blood.

Light-Activated Electron Transfer and Turnover in Ru-Modified Aldehyde Deformylating Oxygenases.

Conversion of biological molecules into fuels or other useful chemicals is an ongoing chemical challenge. One class of enzymes that has received attention for such applications is aldehyde deformylating oxygenase (ADO) enzymes. These enzymes convert aliphatic aldehydes to the alkanes and formate. In this work, we prepared and investigated ADO enzymes modified with RuII(tris-diimine) photosensitizers as a starting point for probing intramolecular electron transfer events. Three variants were prepared, with RuII-modification at the wild type (WT) residue C70, at the R62C site in one mutant ADO, and at both C62 and C70 in a second mutant ADO protein. The single-site modification of WT ADO at C70 using a cysteine-reactive label is an important observation and opens a way forward for new studies of electron flow, mechanism, and redox catalysis in ADO. These Ru-ADO constructs can perform the ADO catalytic cycle in the presence of light and a sacrificial reductant. In this work, the Ru photosensitizer serves as a tethered, artificial reductase that promotes turnover of aldehyde substrates with different carbon chain lengths. Peroxide side products were detected for shorter chain aldehydes, concomitant with less productive turnover. Analysis using semiclassical electron transfer theory supports proposals for hopping pathway for electron flow in WT ADO and in our new Ru-ADO proteins.

Using an artificial tryptophan "wire" in cytochrome c peroxidase for oxidation of organic substrates.

Lignolytic peroxidases use an electron transfer (ET) pathway that involves amino acid-mediated substrate oxidation at the surface of the protein rather than at an embedded heme site. In many of these peroxidases, redox catalysis takes place at a substrate accessible tyrosine or tryptophan (Trp) amino acid. Here, we describe new mutants of cytochrome c peroxidase (CcP) that were designed to incorporate a Trp-based "wire" that can move oxidizing equivalents from the heme to the protein surface. Three mutant CcP proteins were expressed and characterized: A193W, Y229W, and A193W/Y229W. These mutants can oxidize veratryl alcohol substrate with turnover numbers greater than wild type CcP using H2O2 as an oxidant. The A193W/Y229W mutant is the most active. However, the reactivity is still less than typical lignin peroxidases at pH 8. The redox reactivity of these proteins is analysed using semiclassical electron transfer theory. An electron hopping mechanism is possible for A193W/Y229W mutant. These data suggest that artificial chains of aromatic amino acids can support hole transfer from embedded sites to protein surfaces for catalytic redox reactions.

Catalytic reduction of dioxygen with modified Thermus thermophilus cytochrome c552.

Efficient catalysis of the oxygen reduction reaction (ORR) is of central importance to function in fuel cells. Metalloproteins, such as laccase (Cu) or cytochrome c oxidase (Cu/Fe-heme) carry out the 4H(+)/4e(-) reduction quite efficiently, but using large, complex protein frameworks. Smaller heme proteins also can carry out ORR, but less efficiently. To gain greater insight into features that promote efficient ORR, we expressed, characterized, and investigated the electrochemical behavior of six new mutants of cytochrome c552 from Thermus thermophilus: V49S/M69A, V49T/M69A, L29D/V49S/M69A, P27A/P28A/L29D/V49S/M69A, and P27A/P28A/L29D/V49T/M69A. Mutation to V49 causes only minor shifts to Fe(III/II) reduction potentials (E°'), but introduction of Ser provides a hydrogen bond donor that slightly enhances oxygen reduction activity. Mutation of L29 to D induces small shifts in heme optical spectra, but not to E°' (within experimental error). Replacement of P27 and P28 with A in both positions induces a -50 mV shift in E°', again with small changes to the optical spectra. Both the optical spectra and reduction potentials have signatures consistent with peroxidase enzymes. The V49S and V49T mutations have the largest impact of ORR catalysis, suggesting that increased electron density at the Fe site does not improve O2 reduction chemistry.