Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity

Abstract

Chitin deacetylases (CDAs) act on chitin polymers and low molecular weight oligomers producing chitosans and chitosan oligosaccharides. Structurally-defined, partially deacetylated chitooligosaccharides produced by enzymatic methods are of current interest as bioactive molecules for a variety of applications. Among Pochonia chlamydosporia (Pc) annotated CDAs, gene pc_2566 was predicted to encode for an extracellular CE4 deacetylase with two CBM18 chitin binding modules. Chitosan formation during nematode egg infection by this nematophagous fungus suggests a role for their CDAs in pathogenicity. The P. chlamydosporia CDA catalytic domain (PcCDA) was expressed in E. coli BL21, recovered from inclusion bodies, and purified by affinity chromatography. It displays deacetylase activity on chitooligosaccharides with a degree of polymerization (DP) larger than 3, generating mono- and di-deacetylated products with a pattern different from those of closely related fungal CDAs. This is the first report of a CDA from a nematophagous fungus. On a DP5 substrate, PcCDA gave a single mono-deacetylated product in the penultimate position from the non-reducing end (ADAAA) which was then transformed into a di-deacetylated product (ADDAA). This novel deacetylation pattern expands our toolbox of specific CDAs for biotechnological applications, and will provide further insights into the determinants of substrate specificity in this family of enzymes.

Molecular Mechanisms of Chitosan Interactions with Fungi and Plants

“…The fungus overexpresses CDA and chitosanase encoding genes in this process [11]. The activity of the catalytic center of a CDA encoded in the P. chlamydosporia genome has been recently confirmed experimentally [12].…”

RNA-seq and metabolome revealed counter effect of chitosan against Botrytis cinerea on grape berries

“…In case of total mapped reads in control Shine-muscat SC1 have higher percentage i.e. 53,045,265 (89.69%) and in comparison within treatments Kyoho KT1 generated 57,982,396 (88.7%) total mapped reads, followed by ST R2, 52,585,277 (90.07%). Moreover 2,727,030 (4.94%) multiple mapped reads showed in SC2, which is higher as compared to all treatments and varieties.…”

Substrate Recognition and Specificity of Chitin Deacetylases and Related Family 4 Carbohydrate Esterases

“…This aromatic residue is highly conserved (MT4, DxxD( W / Y ), with few exceptions. CE4 enzymes having this aromatic residue seem to prefer a sugar bound in the +1 subsite, and do not deacetylate the reducing end of their substrates, as shown for the Ar CE4 enzyme, as well as for Pes CDA [ 89 ] and Pc CDA [ 95 ], or the reducing end is the slowest position to be deacetylated, as shown for Cl CDA [ 49 ] and An CDA [ 12 ]. On the contrary, Vc CDA has the equivalent aromatic residue in a slightly different position after a two-amino acid insertion in the MT4 motif, and it is part of the Loop 4 that moves from an open to a closed conformation upon substrate binding.…”

“…Pc CDA deacetylates chitooligosaccharides, requiring at least four GlcNAc units in order to be active, but it prefers longer substrates. For DP4 and DP5 substrates, it first deacetylates the penultimate residue from the non-reducing end, and continues to the next residue towards the reducing end, with a pattern of acetylation [ADDA n−3 ] [ 95 ].…”