Structure and function of a CE4 deacetylase isolated from a marine environment

Abstract

Chitin, a polymer of β(1–4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family.

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

“…Few CDAs have been biochemically characterized and only four crystallographic structures have been reported, those from Colletotrichum lindemuthianum (first structure of a CDA) 34 , Aspergillus nidulans 32 , Vibrio cholera 28 (and its ortholog from V. parahaemolyticus ) 43 , and just recently, from a marine Arthrobacter species 44 . Efforts are directed to characterize novel CDAs elucidating the determinants of their activity, specificity and deacetylation pattern, and to use them as biocatalysts for the preparation of pure chitosan oligosaccharides with defined structures.…”

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

“…Arthrobacter species are Gram-positive bacteria known to grow on chitin and secrete chitinases [ 111 , 112 , 113 ]. ArCE4 was identified from a search of monodomain and extracellular deacetylases in annotated genomes and metagenomes [ 64 ]. Ar CE4 is active on α- and β-chitin, chitosan (DA 64%), and acetylxylan.…”

“…The first deacelylation happens at all three internal positions, whereas di-deacetylation mainly occurs at the GlcNAc unit next to the reducing end, and at either of the two other internal units (ADDAA and ADADA). Although other minor products are formed, it seems that the reducing end unit is not deacetylated [ 64 ].…”

“…A second X-ray structure of a CDA in complex with substrate has been recently reported (November 2017), that of Ar CE4 from a marine Arthobacter species [ 64 ]. Even though the enzyme was co-crystallized with a DP4 substrate, only the electron density for a GlcNAc dimer occupying subsite 0 and +1 could be solved.…”

“…The reducing end of the substrate is not deacetylated, or it is the least reactive GlcNAc unit for most CDAs and related CE4 enzymes active on COS. In the 3D structure of the Ar CE4·DP2 complex [ 64 ], binding of the GlcNAc unit of the substrate in the +1 subsite seems to be dominated by the stacking interaction with a Trp in Motif 4. This aromatic residue is highly conserved (MT4, DxxD( W / Y ), with few exceptions.…”