Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product

Abstract

The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the b-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc 4 , GlcNAc 5 and GlcNAc 6 indicated that the predominant reaction involved hydrolysis of GlcNAc 2 from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).

Chitinases Are Essential for Cell Separation in Ustilago maydis

“…Modification of chitin in this compartment might alter the cell wall composition so that filaments do not aggregate and sediment in culture. Such chitin degradation in remnant cell wall material might be similar to autolytic processes observed for ChiB in A. nidulans and ChiB1 in A. fumigatus (65,66). Taking these findings together, U. maydis maintains the expression of one nonconventionally secreted chitinase during its filamentous growth stage, even though its activity is dispensable for morphogenesis.…”

Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis

“…2) (8,10,13,(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). The sequences share several highly conserved fragments among all chitinases in which three regions are important.…”

“…One structural feature, such as that of chitinase B1 from Aspergillus fumigatus (8), contains an extra chitin insertion domain (CID) (9) that comprises ϳ70 -90 amino acids and inserts into the TIM-barrel of CaD. The CID provides residues interacting with a substrate and increases the depth of the substrate binding cleft.…”

A Genetically Engineered Live Attenuated Vaccine ofCoccidioides posadasiiProtects BALB/c Mice against Coccidioidomycosis

“…Results of phylogenetic analyses of the subfamily of Coccidioides chitinases suggested that Cts2, -3, and -4 were most closely related. Two classes of fungal chitinases have been proposed based upon their structural similarity to family 18 chitinases of plants or bacteria (24). The plantlike fungal chitinolytic enzymes of Coccidioides include Cts2, -3, and -4 (25), which are suggested to function as endochitinases that cleave the chitin chain randomly (44).…”