Engineering of a chitin deacetylase to generate tailor-made chitosan polymers.

Chitin deacetylases (CDAs) emerge as a valuable tool to produce chitosans with a nonrandom distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units. We hypothesized before that CDAs tend to bind certain sequences within the substrate matching their subsite preferences for either GlcNAc or GlcN units. Thus, they deacetylate or N-acetylate their substrates at nonrandom positions. To understand the molecular basis of these preferences, we analyzed the binding site of a CDA from Pestalotiopsis sp. (PesCDA) using a detailed activity screening of a site-saturation mutagenesis library. In addition, molecular dynamics simulations were conducted to get an in-depth view of crucial interactions along the binding site. Besides elucidating the function of several amino acids, we were able to show that only 3 residues are responsible for the highly specific binding of PesCDA to oligomeric substrates. The preference to bind a GlcNAc unit at subsite -2 and -1 can mainly be attributed to N75 and H199, respectively. Whereas an exchange of N75 at subsite -2 eliminates enzyme activity, H199 can be substituted with tyrosine to increase the GlcN acceptance at subsite -1. This change in substrate preference not only increases enzyme activity on certain substrates and changes composition of oligomeric products but also significantly changes the pattern of acetylation (PA) when N-acetylating polyglucosamine. Consequently, we could clearly show how subsite preferences influence the PA of chitosans produced with CDAs.

Unique subsite specificity and potential natural function of a chitosan deacetylase from the human pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that infects ∼280,000 people every year, causing >180,000 deaths. The human immune system recognizes chitin as one of the major cell-wall components of invading fungi, but C. neoformans can circumvent this immunosurveillance mechanism by instead exposing chitosan, the partly or fully deacetylated form of chitin. The natural production of chitosans involves the sequential action of chitin synthases (CHSs) and chitin deacetylases (CDAs). C. neoformans expresses four putative CDAs, three of which have been confirmed as functional enzymes that act on chitin in the cell wall. The fourth (CnCda4/Fpd1) is a secreted enzyme with exceptional specificity for d-glucosamine at its -1 subsite, thus preferring chitosan over chitin as a substrate. We used site-specific mutagenesis to reduce the subsite specificity of CnCda4 by converting an atypical isoleucine residue in a flexible loop region to the bulkier or charged residues tyrosine, histidine, and glutamic acid. We also investigated the effect of CnCda4 deacetylation products on human peripheral blood-derived macrophages, leading to a model explaining the function of CnCda4 during infection. We propose that CnCda4 is used for the further deacetylation of chitosans already exposed on the C. neoformans cell wall (originally produced by CnChs3 and CnCda1 to 3) or released from the cell wall as elicitors by human chitinases, thus making the fungus less susceptible to host immunosurveillance. The absence of CnCda4 during infection could therefore promote the faster recognition and elimination of this pathogen.

In silico and in vitro analysis of an Aspergillus niger chitin deacetylase to decipher its subsite sugar preferences.

Chitin deacetylases (CDAs) are found in many different organisms ranging from marine bacteria to fungi and insects. These enzymes catalyze the removal of acetyl groups from chitinous substrates generating various chitosans, linear copolymers consisting of N-acetylglucosamine (GlcNAc) and glucosamine. CDAs influence the degree of acetylation of chitosans as well as their pattern of acetylation, a parameter that was recently shown to influence the physicochemical properties and biological activities of chitosans. The binding site of CDAs typically consists of around four subsites, each accommodating a single sugar unit of the substrate. It has been hypothesized that the subsite preferences for GlcNAc or glucosamine units play a crucial role in the acetylation pattern they generate, but so far, this characteristic was largely ignored and still lacks structural data on the involved residues. Here, we determined the crystal structure of an Aspergillus niger CDA. Then, we used molecular dynamics simulations, backed up with a variety of in vitro activity assays using different well-defined polymeric and oligomeric substrates, to study this CDA in detail. We found that Aspergillus niger CDA strongly prefers a GlcNAc sugar unit at its -1 subsite and shows a weak GlcNAc preference at the other noncatalytic subsites, which was apparent both when deacetylating and N-acetylating oligomeric substrates. Overall, our results show that the combination of in vitro and in silico methods used here enables the detailed analysis of CDAs, including their subsite preferences, which could influence their substrate targets and the characteristics of chitosans produced by these species.

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz - role of cis-prenyltransferase-like 1 protein.

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high-molecular-weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis-1,4-isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis-prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT-like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis-1,4-isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1-RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low-expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS-based comparison of latex proteomes from TkCPTL1-RNAi plants and T. koksaghyz wild-types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.

Preparation of Defined Chitosan Oligosaccharides Using Chitin Deacetylases.

During the past decade, detailed studies using well-defined 'second generation' chitosans have amply proved that both their material properties and their biological activities are dependent on their molecular structure, in particular on their degree of polymerisation (DP) and their fraction of acetylation (FA). Recent evidence suggests that the pattern of acetylation (PA), i.e., the sequence of acetylated and non-acetylated residues along the linear polymer, is equally important, but chitosan polymers with defined, non-random PA are not yet available. One way in which the PA will influence the bioactivities of chitosan polymers is their enzymatic degradation by sequence-dependent chitosan hydrolases present in the target tissues. The PA of the polymer substrates in conjunction with the subsite preferences of the hydrolases determine the type of oligomeric products and the kinetics of their production and further degradation. Thus, the bioactivities of chitosan polymers will at least in part be carried by the chitosan oligomers produced from them, possibly through their interaction with pattern recognition receptors in target cells. In contrast to polymers, partially acetylated chitosan oligosaccharides (paCOS) can be fully characterised concerning their DP, FA, and PA, and chitin deacetylases (CDAs) with different and known regio-selectivities are currently emerging as efficient tools to produce fully defined paCOS in quantities sufficient to probe their bioactivities. In this review, we describe the current state of the art on how CDAs can be used in forward and reverse mode to produce all of the possible paCOS dimers, trimers, and tetramers, most of the pentamers and many of the hexamers. In addition, we describe the biotechnological production of the required fully acetylated and fully deacetylated oligomer substrates, as well as the purification and characterisation of the paCOS products.

Chitosan and Chitin Deacetylase Activity Are Necessary for Development and Virulence of Ustilago maydis.

The biotrophic fungus Ustilago maydis harbors a chitin deacetylase (CDA) family of six active genes as well as one pseudogene which are differentially expressed during colonization. This includes one secreted soluble CDA (Cda4) and five putatively glycosylphosphatidylinositol (GPI)-anchored CDAs, of which Cda7 belongs to a new class of fungal CDAs. Here, we provide a comprehensive functional study of the entire family. While budding cells of U. maydis showed a discrete pattern of chitosan staining, biotrophic hyphae appeared surrounded by a chitosan layer. We purified all six active CDAs and show their activity on different chitin substrates. Single as well as multiple cda mutants were generated and revealed a virulence defect for mutants lacking cda7 We implicated cda4 in production of the chitosan layer surrounding biotrophic hyphae and demonstrated that the loss of this layer does not reduce virulence. By combining different cda mutations, we detected redundancy as well as specific functions for certain CDAs. Specifically, certain combinations of mutations significantly affected virulence concomitantly with reduced adherence, appressorium formation, penetration, and activation of plant defenses. Attempts to inactivate all seven cda genes simultaneously were unsuccessful, and induced depletion of cda2 in a background lacking the other six cda genes illustrated an essential role of chitosan for cell wall integrity.IMPORTANCE The basidiomycete Ustilago maydis causes smut disease in maize, causing substantial losses in world corn production. This nonobligate pathogen penetrates the plant cell wall with the help of appressoria and then establishes an extensive biotrophic interaction, where the hyphae are tightly encased by the plant plasma membrane. For successful invasion and development in plant tissue, recognition of conserved fungal cell wall components such as chitin by the plant immune system needs to be avoided or suppressed. One strategy to achieve this lies in the modification of chitin to chitosan by chitin deacetylases (CDAs). U. maydis has seven cda genes. This study reveals discrete as well as redundant contributions of these genes to virulence as well as to cell wall integrity. Unexpectedly, the inactivation of all seven genes is not tolerated, revealing an essential role of chitosan for viability.