The primary structure of a fungal chitin deacetylase reveals the function for two bacterial gene products.

Abstract

Chitin deacetylase (EC 3.5.1.41) hydrolyzes the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin. A cDNA to the Mucor rouxu mRNA encoding chitin deacetylase was isolated, characterized, and sequenced. Protein sequence comparisons revealed significant imilarities of the fungal chitin deacetylase to rhizobial nodB proteins and to an uncharacterized protein encoded by a Bacillus stearothermophilus open reading frame. These data suggest the functional homology of these evolutionarily distant proteins. NodB is a chitooligosaccharide deacetylase essential for the biosynthesis of the bacterial nodulation signals, termed Nod factors. The observed similarity of chitin deacetylase to the B. stearothermophius gene product suggests that this gene encodes a polysaccharide deacetylase.Chitin exists in the cell wall of several Zygomycetes species in its deacetylated form, referred to as chitosan (1). A chitosan layer has also been identified in the spore wall of Saccharomyces cerevisiae (2). The biosynthesis of chitosan in fungi proceeds by the coordinated action of chitin synthase (EC 2.4.1.16) and chitin deacetylase (EC 3.5.1.41). The former enzyme synthesizes chitin by polymerization of N-acetylglucosaminyl residues from UDP-N-acetylglucosamine, whereas the latter hydrolyzes the N-acetamido groups on the nascent chitin chains (3,4).Chitosan is a biopolymer with unique properties favorable for a broad variety of industrial and biomedical applications (5-7). Presently, chitosan is produced by the thermochemical deacetylation of crab chitin. To develop an alternative enzymatic process for chitosan production, we have initiated a study of fungal chitin deacetylases.Chitin deacetylase from the fungus Mucor rouxii has been purified to homogeneity (8). The enzyme is an acidic glycoprotein of =75 kDa with =30% (wt/wt) carbohydrate content. Further biochemical characterization revealed that the enzyme has a very stringent speciflcity for (31-4-linked N-acetylglucosamine homopolymers, requires at least four residues (chitotetraose) for catalysis, and can achieve extensive deacetylation on chitin polymers.We report here the cloning of a cDNA that encodes M. the AZAP vector, and the packaging extracts were bought from Stratagene. The Sequenase 2.0 kit was purchased from United States Biochemical. Immobilon-P and nitrocellulose membranes were purchased from Millipore. Restriction enzymes were obtained from Minotech. Synthetic oligonucleotides were obtained from the Institute of Molecular Biology and Biotechnology Microchemistry Facility.Growth of M. rouxiu Mycelia. M. rouxii was grown with vigorous shaking at 28°C in a medium containing 0.3% yeast extract, 1% peptone, and 2% glucose; the pH was adjusted to 4.5 with H2SO4 (1). The medium was inoculated with 108 spores per liter, and the mycelia were harvested at early growth phase by fitration.Enzyme Purification. Chitin deacetylase was purified to homogeneity from mycelial extracts of the fungus M. rouxii as described (8).Protein Sequencing. The amino-terminal seq...

Yeast ascospore wall assembly requires two chitin deacetylase isozymes

“…In vitro experiments suggested di¡erent speci¢c-ity of chitin deacetylases on chitin oligosaccharides [13,14]. CDA from the fungus M. rouxii [14], which is inhibited by acetic acid [3], is able to deacetylate chitin oligosaccharides with a degree of polymerization higher than 3 and it fully deacetylates only (GlnNAc) 4 and (GlnNAc) 5 . C. lindemuthianum CDA, on the other hand, which is not inhibited by acetic acid [4], is able to deacetylate (GlnNAc) 2 and fully deacetylates (GlnNAc) 3 to (GlnNAc) 5 [13].…”

“…[1,2] Chitin deacetylase (CDA) is the enzyme that catalyzes the conversion of chitin to chitosan by deacetylation of N-acetyl-D-glucosamine residues. So far, CDA enzymes have been isolated from the fungi Mucor rouxii and Colletotrichum lindemuthianum [3,4] and genes encoding CDA have been studied in M. rouxii and Saccharomyces cerevisiae [5,6].…”

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases

“…CE4 family enzymes are characterised by their dependence, for catalytic activity (deacetylation), on coordination of either a Co 2+ or Zn 2+ cation [68], and by the use of the His-His-Asp metal binding triad [38]. CE4 AcXEs operate an acid (aspartic acid) -base (histidine) catalytic mechanism and are also known as 'NodB homologs' [69][70][71]. They possess the highly conserved catalytic NodB domain [72] characteristic of other rhizobial NodB enzymes in the CE4 family such as the chitin deacetylases, chitooligosaccharide deacetylases, rhizobial nod factor deacetylases as well as peptidoglycan N-acetylglucosamine deacetylases and Nacetylmuramic deacetylases which de-esterify N-or O-acetyl bonds of plant cell wall polymers or oligomers [73][74][75].…”

Mode of action of chitin deacetylase from Mucor rouxii on N‐acetylchitooligosaccharides

“…2A). Peak 3, which corresponds to fully acetylated (GlcNAc) 3 , and peaks a, b that probably correspond to GlcNGlcNAcGlcNAc and (GlcN) 2 GlcNAc, respectively. When incubation of (GlcNAc) 3 with chitin deacetylase was followed by b-N-GlcNAcase treatment prior the Fig.…”

“…Furthermore, a cDNA of the M. rouxii encoding chitin deacetylase was isolated, sequenced and further characterized [3]. Protein sequence comparisons revealed significant similarities between the fungal chitin deacetylase and the rhizobial nodB proteins, suggesting functional homology of these evolutionary distinct proteins.…”