Structural Insights into Cellulolytic and Chitinolytic Enzymes Revealing Crucial Residues of Insect β-N-acetyl-D-hexosaminidase

Abstract

The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind β-1,4-linked glucose residues in a similar manner. A structural and mutational analysis was performed for the plant cellulolytic enzyme BGlu1 from Oryza sativa and the insect chitinolytic enzyme OfHex1 from Ostrinia furnacalis. Although BGlu1 shows little amino-acid sequence or topological similarity with OfHex1, three residues (Trp490, Glu328, Val327 in OfHex1, and Trp358, Tyr131 and Ile179 in BGlu1) were identified as being conserved in the +1 sugar binding site. OfHex1 Glu328 together with Trp490 was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in K m for (GlcNAc)2 and a 42-fold increment in K i for TMG-chitotriomycin. A crystal structure of E328A in complex with TMG-chitotriomycin was resolved at 2.5 Å, revealing the obvious conformational changes of the catalytic residues (Glu368 and Asp367) and the absence of the hydrogen bond between E328A and the C3-OH of the +1 sugar. V327G exhibited the same activity as the wild-type, but acquired the ability to efficiently hydrolyse β-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu328 and Val327 were identified as important for substrate-binding and as glycosidic-bond determinants. A structure-based sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes.

Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications

“…A comparison of the amino acid sequences of chitinases reveals two highly conserved motifs in the family of GH18 chitinases: D XX D X D X E and S X GG, corresponding to the catalytic domains and substrate-binding sites, respectively (Henrissat and Bairoch 1996). Glu (E) and Asp (D) are highly conserved within the catalytic domains of chitinases, indicating their direct involvement in the hydrolysis of glycosidic bond (Li and Greene 2010;Liu et al 2012).…”

Sex-specific association between infant diet and white matter integrity in 8-y-old children

“…FA measurements have been used to identify white matter pathology not evident on conventional MRI (22,23). Furthermore, a recently developed DTI analysis method, tract-based spatial analysis (TBSS), can provide non-subjective voxel-wise analysis of FA values for the whole brain (24), and has shown great sensitivity to white matter abnormalities (25,26).…”

Exploring NAG‐thiazoline and its derivatives as inhibitors of chitinolytic β‐acetylglucosaminidases

“…S7A). The structure‐equivalent residues in SmChb and SpHex to E328 in OfHex1 were Q494 and G277, respectively [51]. Because a glutamine instead of a glycine could form a hydrogen bond similarly to a glutamate, the higher inhibitory activity of NMAGT against SmChb ( K i = 0.23 ± 0.04 μM) than against SpHex ( K i = 20 ± 2 μM) also suggested the importance of E328 (Table 1).…”

“…4C and Table S3. The most obvious difference was related to the residue E328, which was previously known as an important residue for stabilizing the GlcNAc at the subsite +1 in OfHex1 [31,51]. E328 formed two hydrogen bonds with NMAGT but did not interact with NGT.…”