Abstract
Chitinases enzymatically hydrolyze chitin, a highly abundant and utilized polymer of N-acetyl-glucosamine. Fungi are a rich source of chitinases; however, the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C) as chitinases previously assigned to the “C” clade are not resolved as distinct from the “A” clade. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the “B” clade. Furthermore, chitin-binding domains (including the LysM domain) do not define specific clades, but instead are found more broadly across clades of chitinases. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade, one B clade, and one formerly classified C clade chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. These results suggest chitinase activity is variable even within subclades and that predictions of functionality require more sophisticated models.
Highlights
Chitin is a (1,4)-β-linked N-acetyl-D-glucosamine (GlcNAc) polymer
We identified 3,888 glycoside hydrolase family 18 32 (GH18) domain-containing proteins in 373 publicly available fungal genomes (Supplemental Data 1) using an HMM search (Eddy 2009). 494 of these proteins contain the
Maximum likelihood phylogenetic analysis using IQTree was used to establish evolutionary relationships among the fungal chitinases and to infer support for clades and subclades defined by the common ancestor of previously classified chitinases, with some slight expansion for closely related but previously unstudied taxa
Summary
Chitin is a (1,4)-β-linked N-acetyl-D-glucosamine (GlcNAc) polymer. As the second most abundant biopolymer after cellulose (Tharanathan and Kittur 2003), chitin and its deacetylated derivative chitosan are abundant sources of organic carbon and nitrogen that have many potential industrial uses, from biomedical to agricultural to water engineering (Ravi Kumar 2000; Zargar et al 2015). There is a great interest in identifying enzymes that efficiently hydrolyze chitin into more soluble mono- and oligomers of GlcNAc. Chitin degrading enzymes have potential applications in the breakdown of the chitinous structures of fungal and arthropod agricultural pests (Hamid et al 2013). Chitinases (E.C 3.2.1.14) are glycosyl hydrolases that are found in a wide range of plants, bacteria, and fungi. For plants and bacteria, which lack chitin, chitinases play roles primarily in defense against fungi and/or arthropods. Chitin is an important structural component of the fungal cell wall, ranging from 0.5%-5% in yeasts to ≥20% in some filamentous fungi (Hartl et al.2012). Yeast-form fungi possess relatively few chitinases (e.g., two in Saccharomyces cerevisiae) compared to the 10-20 chitinases encoded in some filamentous fungal genomes (Seidl 2008). The expansion of chitinases in filamentous fungi has prompted investigation of chitinase roles in formation of hyphal structures (Seidl 2008). There is evidence that specific chitinases facilitate mycoparasitism of other fungi by Trichoderma (Boer et al 2007; Cruz et al 1992; Seidl et al 2005)
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