The plant cell-wall is constituted by structurally diverse polysaccharides. The biodegradation of these is a crucial process for life sustainability. Cellulolytic microorganisms are highly efficient in this process by assembling modular architectures of carbohydrate-active enzymes with appended non-catalytic carbohydrate-binding modules (CBMs). Carbohydrate microarrays offer high-throughput and sensitive tools for uncovering carbohydrate-binding specificities of CBMs{,} which is pivotal to understand the function of these modules in polysaccharide biodegradation mechanisms. Features of this technology will be here briefly reviewed with highlights of microarray approaches to study plant-carbohydrates and CBM-carbohydrate interactions{,} along with an overview of plant polysaccharides and microorganisms strategies for their recognition.

Understanding the specific molecular interactions between proteins and β1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3-1,4-mixed-linked over β1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the β1,3-linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3-1,4-mixed-linked glucans. This is mediated by a conformation–selection mechanism of the ligand in the binding cleft through CH-π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3-linkage at the reducing end and at specific positions along the β1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked β-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. Database Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

Gold nanoparticles (AuNPs) exhibit unique properties that have made them a very attractive material for application in biological assays. Given the potentially interesting interactions between AuNPs and biological macromolecules, we investigated AuNPs-induced protein crystal growth. Differently functionalized AuNPs were tested as additives in cocrystallization studies with model proteins (hen egg white lysozyme (HEWL), ribonuclease A (RNase A), and proteinase K) as well as with case studies where there were problems in obtaining well-diffracting crystals. Trials were performed considering different crystallization drawbacks, from total absence of crystals to improvement of crystal morphology, size, twinning, and number of crystals per drop. Improvement of some of these factors was observed in the cases of HEWL, RNase A, phenylalanine hydroxylase (PAR), myoglobin, native aldehyde oxidase (AOH), and human albumin. In these proteins, the presence of the AuNPs promoted an increase in the size and/or better crystal morphology. From the systematic trials and subsequent observations, it can be concluded that the introduction of AuNPs should definitely be considered in crystal optimization trials to improve previously determined crystallization conditions.

Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. Mammals are characterized by a complement of species-specific \{AOX\} isoenzymes, that varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, \{AOX3\} and AOX4). The physiological function of mammalian \{AOX\} isoenzymes is unknown, although human \{AOX1\} is an emerging enzyme in phase-I drug metabolism. Indeed, the number of therapeutic molecules under development which act as \{AOX\} substrates is increasing. The recent crystallization and structure determination of human \{AOX1\} as well as mouse \{AOX3\} has brought new insights into the mechanisms underlying substrate/inhibitor binding as well as the catalytic activity of this class of enzymes.