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

Abstract

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.

Preparation of Defined Chitosan Oligosaccharides Using Chitin Deacetylases

“…This will enable us to make use of nature’s biodiversity in our attempts to provide full sets of fully defined paCOS. At the same time, our increasing knowledge of the structures of CDA enzymes, their subsite specificities or preferences, and their enzymatic modes of action will allow us to design and engineer CDA enzymes with new or more precise regio-selectivities to increase and complement the natural biodiversity [ 27 ]. In parallel to these developments, strategies for the chemical synthesis of defined paCOS are also improving, and solid-state synthesis is promising to yield paCOS of every structure at will, even for higher DP, possibly up to DP 10 or even higher [ 80 ].…”

“…They belong to carbohydrate esterase family CE4, and their three-dimensional structures as well as the mode of their catalytic reaction have been described in several recent reviews [ 19 , 20 , 21 , 22 ]. Like many other polysaccharide modifying enzymes, such as chitinases and chitosanases [ 16 , 17 , 23 , 24 , 25 ], CDAs possess a substrate binding site consisting of several subsites, each of which binds a single monosaccharide unit of the substrate [ 26 , 27 , 28 ]. The catalytic subsite, i.e., the one binding the GlcNAc unit that will be deacetylated to yield a GlcN unit, is defined as subsite {0}, the subsite(s) ‘left’ of it, i.e., towards the non-reducing end of the substrate, are numbered as {−1}, {−2}, etc., while those ‘right’ of subsite {0}, i.e., towards the reducing end of the substrates, are {+1} etc.…”

“…A partial or full negative charge in the substrate binding cleft may favour, a positive one disfavour the binding of a GlcN unit at that particular position, while the binding of a GlcNAc unit can be expected to require more space and to be supported by the presence of a hydrophobic pocket to accommodate the terminal methyl group of the acetamido side chain. In fact, the first chitosan (rather than chitin) deacetylase has recently been described, which favours GlcN over GlcNAc at subsite {−1}, and thus partially acetylated chitosan over chitin as a substrate [ 27 ].…”

“…The chitosan deacetylase CnCDA4, for example, starts de-acetylating the fully acetylated pentamer A5 at the position next to the reducing end of the substrate, yielding AAADA. However, as the enzyme highly prefers a de-acetylated GlcN unit at subsite {−1}, it is impossible to predict whether it would produce ADADA or ADDAA when acting on ADAAA as a substrate [ 27 ]. Thus, when targeting the paCOS ADADA, it is preferable to first use CnCDA4 on A5 yielding AAADA followed by VcCDA to yield the targeted ADADA, rather than vice versa.…”

“…Let us look at the production of AADAAA. As described by Hembach et al (2020) [ 27 ], ADDAAA can be produced from A6 by the sequential action of the mutein VcCDA K275E/H127 yielding ADAAAA followed by CnCDA4 (or possibly directly by PcCDA acting on A6). Then, ADDAAA needs to be N -acetylated again using the VcCDA mutein to produce the targeted AADAAA.…”