Characterization and crystal structure of a thermostable glycoside hydrolase family 45 1,4-β-endoglucanase from Thielavia terrestris

Gao, Jian; Huang, Jian‐Wen; Li, Qian; Liu, Weidong; Ko, Tzu‐Ping; Zheng, Yingying; Xiao, Xiansha; Kuo, C.J.; Chen, Chun‐Chi; Guo, Rey‐Ting

doi:10.1016/j.enzmictec.2017.01.005

Cited by 25 publications

(26 citation statements)

References 19 publications

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“…To improve specific activity and thermostability of the C. thermophilum endoglucanase CTendo45, rational protein engineering was carried out in this study. Thielavia terrestris β-1,4-endoglucanase TtCel45A (PDB: 5GLY) in complex with cellotriose and cellotetraose, carrying 64% amino-acid identity with CTendo45, was used as the basis for a homology model to predict the structure of candidate mutations 24 . As a typical GH45 endoglucanase 6 , 25 , TtCel45A exhibits the characteristic six-stranded β-barrel and a region consisting of several long interconnecting loops, which are partitioned by a substrate-binding cleft spanning the protein surface (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…They are positioned above and to either side of a noncatalytic residue, Y10, which lies at the bottom of the active side groove 6 . The structural model also indicates the importance of Y148 based on its aromatic residue at the +1 subsite 24 , 26 . Detailed substrate interaction networks demonstrated that the main chains of R9 and W11 participate in the formation of subsites −1, +1 and +2 27 , 28 .…”

Section: Resultsmentioning

confidence: 99%

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Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability

Chen

et al. 2018

Sci Rep

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Endoglucanases are increasingly applied in agricultural and industrial applications as a key biocatalyst for cellulose biodegradation. However, the low performance in extreme conditions seriously challenges the enzyme’s commercial utilization. To obtain endoglucanases with substantially improved activity and thermostability, structure-based rational design was carried out based on the Chaetomium thermophilum β-1,4-endoglucanase CTendo45. In this study, five mutant enzymes were constructed by substitution of conserved and noncatalytic residues using site-directed mutagenesis. Mutants were constitutively expressed in Pichia pastoris, purified, and ultimately tested for enzymatic characteristics. Two single mutants, Y30F and Y173F, increased the enzyme’s specific activity 1.35- and 1.87-fold using carboxymethylcellulose sodium (CMC-Na) as a substrate, respectively. Furthermore, CTendo45 and mutants exhibited higher activity towards β-D-glucan than that of CMC-Na, and activities of Y173F and Y30F were also increased obviously against β-D-glucan. In addition, Y173F significantly improved the enzyme’s heat resistance at 80 °C and 90 °C. More interestingly, the double mutant Y30F/Y173F obtained considerably higher stability at elevated temperatures but failed to inherit the increased catalytic efficiency of its single mutant counterparts. This work gives an initial insight into the biological function of conserved and noncatalytic residues of thermostable endoglucanases and proposes a feasible path for the improvement of enzyme redesign proposals.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability

Chen

et al. 2018

Sci Rep

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“…enzymes. In the case of the subfamily A enzymes the sugar unit at subsite À1 should be bound in a much more distorted conformation, as found in the complex of Theilavia terrestris Cel45A with cellotetraose (Gao et al, 2017). The subsite À1 sugar of -glycosidases, including cellulases, must adopt a distorted conformation during the direct inline nucleophilic attack (Davies et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

“…It has been reported that endoglucanases in subfamily A have a glycine-rich loop called the 'V-VI loop' between the 5 and 6 strands, which shows a large movement when the oligosaccharides bind to the enzyme (Davies et al, 1995;Gao et al, 2017;Song et al, 2017). It has been presumed that the role of the V-VI loop is either to prevent nucleophilic molecules other than water from entering the catalytic site (Davies et al, 1995) or to move the catalytic acid to the correct position (Gao et al, 2017). Since subfamily B and C endoglucanases do not conserve the amino-acid sequence of the V-VI loop of subfamily A (it is not glycine-rich), the V-VI loops of EG27II and PcCel45A have a completely different structure from that of subfamily A ('V-VI Loop' in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, fungal cellulases are distributed in all subfamilies. Structural analysis of several enzymes in subfamily A has been performed (Davies et al, 1993(Davies et al, , 1995Hirvonen & Papageorgiou, 2003;Valjakka & Rouvinen, 2003;Gao et al, 2017;Song et al, 2017). Based on these studies, it was presumed that GH45 enzymes catalyze via an inverting mechanism with two aspartic acid residues acting as the catalytic residues (Davies et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

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High-resolution crystal structures of the glycoside hydrolase family 45 endoglucanase EG27II from the snailAmpullaria crossean

Nomura

Iwase

Saka

et al. 2019

Acta Cryst Sect D Struct Biol

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Although endogenous animal cellulases have great potential for industrial applications such as bioethanol production, few investigations have focused on these enzymes. In this study, the glycoside hydrolase family 45 (GH45) subfamily B endoglucanase EG27II from the snail Ampullaria crossean was expressed using a Pichia pastoris expression system and the crystal structure of the apo form was determined at 1.00 Å resolution; this is the highest resolution structure of an animal endoglucanase. The results showed that EG27II has a double‐ψ six‐stranded β‐barrel and that the structure of EG27II more closely resembles those of subfamily C enzymes than those of subfamily A enzymes. The structure of EG27II complexed with cellobiose was also determined under cryoconditions and at room temperature at three pH values, pH 4.0, 5.5 and 8.0, and no structural changes were found to be associated with the change in pH. The structural comparison and catalytic activity measurements showed that Asp137 and Asn112 function as the catalytic acid and base, respectively, and that Asp27 is also an important residue for catalysis. These high‐resolution structures of EG27II provide a large amount of information for structure‐based enzyme modification and cell‐surface engineering, which will advance biofuel production using animal‐derived cellulases.

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Cellulases from Thermophilic Fungi: Recent Insights and Biotechnological Potential

Papageorgiou

2019

Fungi in Extreme Environments: Ecological Role and Biotechnological Significance

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Characterization and crystal structure of a thermostable glycoside hydrolase family 45 1,4-β-endoglucanase from Thielavia terrestris

Cited by 25 publications

References 19 publications

Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability

Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability

High-resolution crystal structures of the glycoside hydrolase family 45 endoglucanase EG27II from the snailAmpullaria crossean

Cellulases from Thermophilic Fungi: Recent Insights and Biotechnological Potential

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