Structural Determinants of an Insect β-N-Acetyl-d-hexosaminidase Specialized as a Chitinolytic Enzyme

Abstract

␤-N-Acetyl-D-hexosaminidase has been postulated to have a specialized function. However, the structural basis of this specialization is not yet established. OfHex1, the enzyme from the Asian corn borer Ostrinia furnacalis (one of the most destructive pests) has previously been reported to function merely in chitin degradation. Here the vital role of OfHex1 during the pupation of O. furnacalis was revealed by RNA interference, and the crystal structures of OfHex1 and OfHex1 complexed with TMG-chitotriomycin were determined at 2.1 Å . The mechanism of selective inhibition by TMG-chitotriomycin was related to the existence of the ؉1 subsite at the active pocket of OfHex1 and a key residue, Trp 490 , at this site. Mutation of Trp 490 to Ala led to a 2,277-fold decrease in sensitivity toward TMG-chitotriomycin as well as an 18-fold decrease in binding affinity for the substrate (GlcNAc) 2 . Although the overall topology of the catalytic domain of OfHex1 shows a high similarity with the human and bacterial enzymes, OfHex1 is distinguished from these enzymes by large conformational changes linked to an "open-close" mechanism at the entrance of the active site, which is characterized by the "lid" residue, Trp 448 . Mutation of Trp 448 to Ala or Phe resulted in a more than 1,000-fold loss in enzyme activity, due mainly to the effect on k cat . The current work has increased our understanding of the structure-function relationship of OfHex1, shedding light on the structural basis that accounts for the specialized function of ␤-N-acetyl-D-hexosaminidase as well as making the development of species-specific pesticides a likely reality.␤-N-Acetyl-D-hexosaminidase (EC 3.2.1.52), a member of the family 20 glycosyl hydrolyases (GH20), 4 is an enzyme that participates in the breakdown of glycosidic bonds of glycans, glycoproteins, and glycolipids (1). It has been postulated to have specialized physiological functions, including post-translational modification of N-glycans, degradation of glycoconjugates, and egg-sperm recognition (1). The structural basis for these specialized functions is still unclear.It is interesting to note that insects have evolved to have more than one ␤-N-acetyl-D-hexosaminidase, as revealed by genomic analysis of various insects, including Coleoptera, Diptera, Hymenoptera, Lepidoptera, Phthiraptera, and Hemiptera. The activities of insect ␤-N-acetyl-D-hexosaminidases are not restricted to chitin degradation but are also associated with post-translational modification of N-glycans, degradation of glycoconjugates, and egg-sperm recognition, suggesting that these enzymes have rather versatile physiological functions in the growth and development of insects (2). Some of these physiological functions may overlap with those of the same enzymes found in higher organisms. Mammal lysosomal ␤-N-acetyl-D-hexosaminidases are mainly responsible for glycoconjugate degradation in lysosome (3). Likewise, ␤-N-acetyl-D-hexosaminidases from the insects Bombyx mori (4) and Spodoptera frugiperda (5) have broad substrate spe...

A crystal structure-guided rational design switching non-carbohydrate inhibitors' specificity between two β-GlcNAcase homologs

“…Structure-based comparison between unliganded and Q2 -complexed OfHex1 reveals that several residues including H303, D367, E368, W448 and E526 undergo large conformational changes in a manner similar to OfHex1 complexed with TMG-chitotriomycin10 (Fig. 4a).…”

A Second β-Hexosaminidase Encoded in the Streptococcus pneumoniae Genome Provides an Expanded Biochemical Ability to Degrade Host Glycans

“…human HexA and HexB); however, usually such enzymes display specificity for one configuration of sugar or the other (9,44,48). GH20C from S. pneumoniae is among only a small number of characterized enzymes that have similar activities on both types of terminal sugar.…”

Crystal Structures of a Glycoside Hydrolase Family 20 Lacto-N-biosidase from Bifidobacterium bifidum

“…The GlcNAc moieties of these substrates bound to the Ϫ1 subsite were all in conformations close to a 1,4 B conformation (229°Ͻ Ͻ 254°and Ͼ 66°; ideally ϭ 240°and ϭ 90°). Moreover, the complex structure of TMG-chitotriomycin, a linear tetrasaccharide inhibitor (55), bound to the ␤-HexNAcase from Ostrinia furnacalis was reported (30) with the N,N,Ntrimethyl-D-glucosamine group at the nonreducing end of TMG-chitotriomycin also adopting a 1,4 B-like conformation ( ϭ 244.2°and ϭ 84.5°). Therefore, the substrate of the Michaelis complex for GH20 enzymes is thought to adopt a 1,4 B conformation.…”