A Novel Glycoside Hydrolase Family 113 Endo-β-1,4-Mannanase from Alicyclobacillus sp. Strain A4 and Insight into the Substrate Recognition and Catalytic Mechanism of This Family

Abstract

c Few members of glycoside hydrolase (GH) family 113 have been characterized, and information on substrate recognition by and the catalytic mechanism of this family is extremely limited. In the present study, a novel endo-␤-1,4-mannanase of GH 113, Man113A, was identified in thermoacidophilic Alicyclobacillus sp. strain A4 and found to exhibit both hydrolytic and transglycosylation activities. The enzyme had a broad substrate spectrum, showed higher activities on glucomannan than on galactomannan, and released mannobiose and mannotriose as the main hydrolysis products after an extended incubation. Compared to the only functionally characterized and structure-resolved counterpart Alicyclobacillus acidocaldarius ManA (AaManA) of GH 113, Man113A showed much higher catalytic efficiency on mannooligosaccharides, in the order mannohexaose Ϸ mannopentaose > mannotetraose > mannotriose, and required at least four sugar units for efficient catalysis. Homology modeling, molecular docking analysis, and site-directed mutagenesis revealed the vital roles of eight residues (Trp13, Asn90, Trp96, Arg97, Tyr196, Trp274, Tyr292, and Cys143) related to substrate recognition by and catalytic mechanism of GH 113. Comparison of the binding pockets and key residues of ␤-mannanases of different families indicated that members of GH 113 and GH 5 have more residues serving as stacking platforms to support ؊4 to ؊1 subsites than those of GH 26 and that the residues preceding the acid/base catalyst are quite different. Taken as a whole, this study elucidates substrate recognition by and the catalytic mechanism of GH 113 ␤-mannanases and distinguishes them from counterparts of other families.A major component of hemicellulose in the plant cell walls of softwood, plant seeds, and beans is ␤-1,4-mannan, including mannan polysaccharides, glucomannan, galactomannan, and galactoglucomannan. It is composed of a backbone of ␤-1,4-linked mannose or a combination of glucose and mannose with side chains of ␣-1,6-linked galactose residues (1). The mannan-degrading enzymes have biotechnological applications in various areas, such as feed manufacturing (2), paper processing (3), and coffee extract treatment (4). Mannooligosaccharides, the major hydrolysis products of mannan, are beneficial as animal nutrition additives due to their potential prebiotic properties (5). The complete degradation of mannan polysaccharides into monomers requires an enzyme system including ␤-mannanase (EC 3.2.1.78), ␤-mannosidase (EC 3.2.1.25), ␤-glucosidase (EC 3.2.1.21), and ␣-galactosidase (EC 3.2.1.22). Of these, ␤-mannanase randomly hydrolyzes the ␤-D-1,4-mannopyranoside linkages and plays the major role in mannan degradation.␤-Mannanases are widely distributed in various organisms, including bacteria, yeasts, filamentous fungi, and plants. Based on the amino acid sequence and structural similarities of catalytic domains, ␤-mannanases are grouped into three glycoside hydrolase (GH) families in the Carbohydrate-Active enZYmes (CAZy) database (6), i.e., 5, 26, and 113. GH 1...

Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 β-1,4-mannanase from Amphibacillus xylanus

“…The catalytic residues are Glu-143 and Glu-223 in AxMan113A, as confirmed by site-directed mutagenesis and structural alignment to AaManA. Previous studies have shown that GH family 113 members hydrolyze mannooligosaccharides with DP Ͼ 3 and exhibit considerable transglycosylation activity (10,19). However, the smallest substrate of AxMan113A was M2 (Fig.…”

“…The specific activity of AxMan113A toward konjac powder was higher than that toward LBG or guar gum, which is consistent with other GH family 113 members, such as AaManA (10) and Man113A from Alicyclobacillus sp. strain A4 (19). In contrast, ␤-1,4-mannanases from GH families 5, 26, and 134, such as RmMan5A from Rhizomucor miehei (20), ReTMan26 from ther- mophilic Bacillus subtilis (21), and AoMan134A from Aspergillus oryzae (22), exhibit high hydrolysis preference for LBG.…”

Mannanase hydrolysis of spruce galactoglucomannan focusing on the influence of acetylation on enzymatic mannan degradation

“…Mannanases release mainly mannobiose and mannotriose as end products, and exhibit open cleft-shaped active sites able to accommodate up to 5–6 hexose units [ 22 , 23 ]. Based on the amino acid sequences, mannanases have been described in three glycoside hydrolase (GH) families in the Carbohydrate-Active enZYmes database (CAZy) [ 24 , 25 ]: GH5, GH26 and recently also GH113 [ 26 ]. The majority of the characterized mannanases have been classified into the GH5 and GH26 families [ 26 , 27 ], which both belong to the glycoside hydrolase clan GH-A.…”

Isolation of Mannanase-producing Bacteria, Bacillus subtilis WL-6 and WL-11, and Cloning and Characterization of Mannanase

“…최근에는 GH113에 속하는 Alicyclobacillus속 균주의 mannanase [19]와 GH134에 속하는 A. nidulans의 mannanase [13]가 보고되었다. 한편 GH5와 GH113에 속하는 mannanase는 GH26의 mannanase와는 달리 당 전이활성을 보이는 것으로 알려졌다 [11,19].…”

“…최근에는 GH113에 속하는 Alicyclobacillus속 균주의 mannanase [19]와 GH134에 속하는 A. nidulans의 mannanase [13]가 보고되었다. 한편 GH5와 GH113에 속하는 mannanase는 GH26의 mannanase와는 달리 당 전이활성을 보이는 것으로 알려졌다 [11,19]. Bacillus속 균주로부터 여러 종류의 mannanases에 대한 반응특성, 구조 및 그 유전자가 밝혀졌으며, 대장균을 비롯하여 B. subtilis, B. brevis [25], Pichia pastoris [8] 등의 다양한 균주를 숙주균으로 하여 Bacillus 유래 의 mannanase를 생산하는 재조합 균주가 개발되었다.…”