Characterization and identification of essential residues of the glycoside hydrolase family 64 laminaripentaose-producing- -1, 3-glucanase

Abstract

Laminaripentaose-producing β-1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of β-1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to >90% homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residues--Asp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.

Novel Anti-Fungal d-Laminaripentaose-Releasing Endo-β-1,3-glucanase with a RICIN-like Domain from Cellulosimicrobium funkei HY-13

“…When the enzyme reactions were conducted in the presence of tryptophan (Trp) residue-specific modifiers including Hg 2+ (1 mM) and N-bromosuccinimide (5 mM), rGluY was considerably inactivated by the compounds (Figure 4). Previously, a similar observation was also made with a GH64 endo-β-1,3-glucanase from S. matensis DIC-108 [6] reacted with curdlan in the presence of Hg 2+ (>20 µM), although the biocatalytic activity of Laceyella putida GH16 endo-β-1,3-glucanase [27] was only moderately suppressed by the metal ion. Taken together, the findings were consistent with the fact that N-bromosuccinimide and Hg 2+ ions oxidize the indole ring of strictly conserved Trp residues in the active site of endotype GH enzymes, which essentially participate in enzyme-substrate interaction [28,29].…”

“…strain YCWD3 [22] and Oerskovia xanthineolytica (Recently Cellulosimicrobium cellulans) [23], which share 99% amino acid sequence identity [24], have been reported to be partially characterized to date. The two conserved residues, Asp169 acting as the catalytic nucleophile/base and Glu153 acting as the catalytic proton donor, were observed in the active site of premature GluY, as shown in other GH64 endo-β-1,3-glucanases [6]. β-1,3-glucanase (WP_083711238); Jsp, Jiangella sp.…”

“…However, the biocatalytic capacity of rGluY was remarkably reduced at pH values below 4.0 (<30% of its maximum activity) or above 8.5 (<65% of its maximum activity), and at temperatures over 50 • C (<65% of its maximum activity). The optimum pH and temperature of rGluY was comparable to a GH64 endo-β-1,3-glucanase from S. matensis DIC-108, which was most active toward curdlan at 55 • C in pH range 7.5-8.5 [6]. Compared to rGluY, rGluY∆RICIN displayed a maximum degradation activity for laminarin at 45 • C and pH 5.5, similar to the GH64 endo-β-1,3-glucanase (KfGH64) from Kribbella flavida NBRC 14,399 [26].…”

“…These values agreed well with the deduced molecular masses of rGluY (57,274 Da) and rGluYΔRICIN (42,783 Da), which were determined by the Compute pI/MW server (https://www.expasy.org/resources/compute-pi-mw). The molecular size (57.5 kDa) of rGluY with a RICIN domain was larger than that (39.4 kDa) of a non-modular GH64 endo-β-1,3-glucanase from Streptomyces matensis DIC-108 [6], that (41.0 kDa) of a non-modular GH64 endo-β-1,3-glucanase from Lysobacter enzymogenes N4-7 [25], and that (43.0 kDa) of a non-modular GH64 endo-β-1,3-glucanase from Kribbella flavida NBRC 14,399 [26] (Table 1). However, the molecular mass (57.5 kDa) of the enzyme was similar to that (55.0 kDa) of a modular GH64 endo-β-1,3-glucanase from Arthrobacter sp.…”

“…However, the majority of endoβ-1,3-glucanases are distributed between four GH families (16, 55, 64, and 81). The GH16 endo-β-1,3-glucanases are retaining enzymes with a β-jelly roll structure, while GH55, GH64, and GH81 endo-β-1,3-glucanases are inverting enzymes [6]. Due to their anti-fungal Biomolecules 2021, 11, 1080 2 of 15 activity against pathogenic fungi, endo-β-1,3-glucanases have attracted a great deal of attention in the food and pharmaceutical industries as potential biocontrol agents [7,8].…”