The <i>Humicola grisea</i> Cel12A enzyme structure at 1.2 Å resolution and the impact of its free cysteine residues on thermal stability

Sandgren, Mats; Gualfetti, Peter; Paech, Christian; Paech, Sigrid; Shaw, Andrew M.; Gross, L.S.; Saldajeno, M.; Berglund, G.I.; Jones, T. Alwyn; Mitchinson, Colin

doi:10.1110/ps.03220403

Cited by 38 publications

(33 citation statements)

References 45 publications

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“…The docking grid size was set to 66 Å by 66 Å by 66 Å, which was placed in an appropriate cavity in the catalytic pocket. The docked complexes were analyzed to select the most reliable binding conformation, as performed in previous studies (4,25). Molecular dynamics (MD) simulations were carried out using the Amber ff99SB force field at a temperature of 338 K (328 K for complexes EG and the EGΔSTTQA mutant with ligands) for 32 ns.…”

Section: Strainsmentioning

confidence: 99%

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Yang

Shi

Liu

et al. 2017

Appl Environ Microbiol

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Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4) from Aspergillus niger. Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (k cat /K m , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (k cat /K m ) of these enzymes (in descending order, NfEG12A ). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (k cat ). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the Ϫ1 or ϩ1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (k cat ). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.KEYWORDS Neosartorya fischeri, GH12 ␤-endoglucanase, loop, catalytic efficiency, hydrogen bond I n recent decades, bioethanol as a clean energy source has become a hot spot of research and industrial application (1). The bioconversion processes of producing fermentable sugars from biomass need a few glycoside hydrolases (GH), especially the

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Section: Strainsmentioning

confidence: 99%

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Yang

Shi

Liu

et al. 2017

Appl Environ Microbiol

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“…Arranged in content order, the two amino acids are located in the third and the twentieth, respectively ( Table 2). Many studies have shown that the number of proline residues is positively correlated with the thermal stability of the enzyme, while the content of cysteine residues decreases with increasing enzyme thermostability [30,31] It is possible that the number of these particular amino acids making up this EGt contributes to its thermostablity. a) Glutamine and asparamide were converted to glutamic acid and aspartic acid, while tryptophan was completely destroyed.…”

Section: Characterization Of Enzymesmentioning

confidence: 99%

A novel thermophilic endoglucanase from a mesophilic fungus Fusarium oxysporum

Liu

Duan

et al. 2006

CHINESE SCI BULL

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A novel thermophilic endoglucanase (EGt) was extracted from a mesophilic fungus (Fusarium oxysporum L19). We invoked conventional kinetic enzyme reactions using the sodium salt of carboxymethyl cellulose (CMC-Na) as substrate. EGt displayed optimal activity at 75℃ when kept running 30 min in the temperature range of 30-85℃. Thermal stability curve measured at 70℃ suggested that its half-life time is 15.1 min. The activity was enhanced in the presence of Co 2+ or Mg 2+ but inhibited by Pb 2+ and Fe 3+ . Moreover, N-bromosuccinimide (NBS) modification resulted in a complete loss of EGt activity, suggesting that tryptophan residues may be involved in the enzyme active site. Amino acid composition analysis demonstrated that EGt contains more proline residues. EGt lacks activity towards p-nitrophenyl cellobiose (pNPC). The N-terminal amino acid sequence of EGt is SYRVPAANGFPNPDASQEKQ, and the gene of EGt was sequenced and analyzed. Extensive sequence alignments failed to show any homology between EGt and any known endoglucanases. This is the first report addressing the thermal adaptation of a cellulolytic enzyme from the mesophilic fungus F. oxysporum. Maybe the expression of multiple isoenzyme in an organism helps it adapt to complex living environments.

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“…These residues are involved in van der Waals interactions. 37) Further, there are several lines of evidence that free cysteine residues contribute to protein thermostability. [38][39][40] On the other hand, there are reports that both buried and exposed cysteine residues increase irreversible unfolding to form incorrect disulfide bonds.…”

Section: )mentioning

confidence: 99%

A Single Free Cysteine Residue and Disulfide Bond Contribute to the Thermostability ofAspergillus saitoi1,2-α-Mannosidase

Tatara

Yoshida

Ichishima

2005

Bioscience, Biotechnology, and Biochemistry

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Aspergillus saitoi 1,2--mannosidase contains three conserved cysteine residues (Cys334, Cys363, and Cys443). We showed that Cys334 and Cys363 are involved in a disulfide bond, and that Cys443 contains a free thiol group. The cysteines were not essential for the activity analyzed by site-directed mutagenesis and kinetics. The substitution at each cysteine residue greatly destabilized the enzyme. The T m values of WT, C443A, C443G, C443S, and C443T were 55.8, 51.9, 50.2, 50.0, and 52.8 C respectively. The specific activity of these mutants was almost equal to that of WT. Introducing Asp, Leu, Met, or Val at position 443 caused partial denaturation, although the enzymes had some activity. C443F, C443I, C443N, and C443Y were not secreted. These results suggest that the hydrophilic and large side chain causes the destabilization. Molecular modelling showed that the Cys443 residue is buried and surrounded by a hydrophobic environment. Cys334 and Cys363 form a disulfide bond, and Cys443 is involved in a hydrophobic interaction to stabilize the enzyme.

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The Humicola grisea Cel12A enzyme structure at 1.2 Å resolution and the impact of its free cysteine residues on thermal stability

Cited by 38 publications

References 45 publications

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

A novel thermophilic endoglucanase from a mesophilic fungus Fusarium oxysporum

A Single Free Cysteine Residue and Disulfide Bond Contribute to the Thermostability ofAspergillus saitoi1,2-α-Mannosidase

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