Crystal and Molecular Structure at 0.16-nm Resolution of the Hybrid Bacillus Endo-1,3-1,4-beta-D-Glucan 4-Glucanohydrolase H(A16-M)

Abstract

H(A16‐M) is a hybrid endo‐1,3‐1,4‐β‐D‐glucan 4‐glucanohydrolase from Bacillus. Its crystal structure was refined using synchrotron X‐ray diffraction data up to a maximal resolution of 0.16 nm. The R value of the resulting model is 14.3% against 21032 reflections >2σ. 93% of the amino acid residues are in the most favorable regions of the Ramachandran diagram, and geometrical parameters are in accordance with other proteins solved at high resolution. As shown earlier [Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Acad. Sci. USA 90, 5287–5291], the protein folds into a compact jellyroll‐type β‐sheet structure. A systematic analysis of the secondary structure reveals the presence of two major antiparallel β‐sheets and a three‐stranded minor mixed sheet. Amino acid residues involved in catalysis and substrate binding are located inside a deep channel spanning the surface of the protein. To investigate the stereochemical cause of the observed specificity of endo‐1,3‐1,4‐β‐D‐glucan 4‐glucanohy‐drolases towards β‐1,4 glycosyl bonds adjacent to β‐1,3 bonds, the high‐resolution crystal structure has been used to model an enzyme‐substrate complex. It is proposed that productive substrate binding to the subsites p1, p2 and p3 of H(A16‐M) requires a β‐1,3 linkage between glucose units bound to p1 and p2.

Crystal structure of Bacillus licheniformis 1,3‐1,4‐β‐d‐glucan 4‐glucanohydrolase at 1.8 Å resolution

“…The overall structure of BGLL is nearly identical to the known structures of the Bacillus 1,3-1,4-fl-glucanases BGLM and H(A16-M) [10,11]. The fold is dominated by two antiparal-M major fl-sheets that consists of 8 and 7 strands, respectively (Fig.…”

“…They show sequence similarities with fl-l,3-glucanases (laminarinases) but not with 1,3-1,4-fl-glucanases from plants [8]. The crystal structure of the 1,3-1,4-fl-glucanase of B. macerans (BGLM) and the closely related hybrid Bacillus enzyme H(A16-M) are known as well as circularly permuted variants thereof [9][10][11]. 1,3-1,4-fl-Glucanases belong to the jellyroll fl-sandwich-type proteins, where the sheets of the sandwich are curved and create a channel for substrate binding and cleavage.…”

Highly thermostable endo-1,3-β -glucanase (laminarinase) Lam A from Thermotoga neapolitana: nucleotide sequence of the gene and characterization of the recombinant gene product

“…Within the amino acid sequence of lamA, three domains (Al-Cat-A2) can be distinguished in addition to a hydrophobic N terminus with the features of a typical signal peptide (van Heijne, 1986) (Fig. 2).…”

“…This possibly reflects the difference in substrate-binding activity towards 1,3-p-or 1,3-1,4-pglucans and in extension of the hydrolytic activity towards 1,3-p-linkages. Surprisingly, B-sheets N and 0, lying within the outside shell of the 'jelly roll' (Hahn et al, 1995), are highly conserved, pointing to a possible crucial role for the structural integrity of the p-glucanase …”

“…According to the molecular model of the hybrid Bacillus 1,3-1,4-P-glucanase, the general acid is Glu107 (Glu343 in LamA) and the catalytic nucleophile is Glu103 (Glu338) (Hahn et al, 1995). These residues are, in addition to Asp105 (Asp340) and GlyllO (Gly346), highly conserved throughout the sequences of bacterial lichenases and laminarinases (Fig.…”