Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme.


Abstract

Several models have been proposed to explain the high temperatures required to denature enzymes from thermophilic organisms; some involve greater maximum thermodynamic stability for the thermophile, and others do not. To test these models, we reversibly melted two analogous protein domains in a two-state manner. E2cd is the isolated catalytic domain of cellulase E2 from the thermophile Thermomonospora fusca. CenAP30 is the analogous domain of the cellulase CenA from the mesophile Cellulomonas fimi. When reversibly denatured in a common buffer, the thermophilic enzyme E2cd had a temperature of melting (Tm) of 72.2 degrees C, a van't Hoff enthalpy of unfolding (DeltaHVH) of 190 kcal/mol, and an entropy of unfolding (DeltaSu) of 0.55 kcal/(mol*K); the mesophilic enzyme CenAP30 had a Tm of 56.4 degrees C, a DeltaHVH of 107 kcal/mol, and a DeltaSu of 0. 32 kcal/(mol*K). The higher DeltaHVH and DeltaSu values for E2cd suggest that its free energy of unfolding (DeltaGu) has a steeper dependence on temperature at the Tm than CenAP30. This result supports models that predict a greater maximum thermodynamic stability for thermophilic enzymes than for their mesophilic counterparts. This was further explored by urea denaturation. Under reducing conditions at 30 degrees C, E2cd had a concentration of melting (Cm) of 5.2 M and a DeltaGu of 11.2 kcal/mol; CenAP30 had a Cm of 2.6 M and a DeltaGu of 4.3 kcal/mol. Under nonreducing conditions, the Cm and DeltaGu of CenAP30 were increased to 4.5 M and 10.8 kcal/mol at 30 degrees C; the Cm for E2cd was increased to at least 7.4 M at 32 degrees C. We were unable to determine a DeltaGu value for E2cd under nonreducing conditions due to problems with reversibility. These data suggest that E2cd attains its greater thermal stability (DeltaTm = 15.8 degrees C) through a greater thermodynamic stability (DeltaDeltaGu = 6.9 kcal/mol) compared to its mesophilic analogue CenAP30. Study holds ProTherm entries: 5614, 5615, 5616, 5617, 5618, 5619 Extra Details: thermodynamic stability; free energies of unfolding;,thermophilic enzyme; mesophilic counterpart

Submission Details

ID: 4hESejnu3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Beadle BM;Baase WA;Wilson DB;Gilkes NR;Shoichet BK,Biochemistry (1999) Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme. PMID:10029552
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1EXH 1995-06-03 SOLUTION STRUCTURE OF A CELLULOSE BINDING DOMAIN FROM CELLULOMONAS FIMI BY NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
1EXG 1995-06-03 SOLUTION STRUCTURE OF A CELLULOSE BINDING DOMAIN FROM CELLULOMONAS FIMI BY NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
2BOG 2005-10-05 1.04 Catalytic domain of endo-1,4-glucanase Cel6A mutant Y73S from Thermobifida fusca in complex with methyl cellobiosyl-4-thio-beta- cellobioside
2BOE 2005-10-05 1.15 Catalytic domain of endo-1,4-glucanase Cel6A mutant Y73S from Thermobifida fusca
3RPT 2011-12-21 1.3 Crystal structure of the anti-HIV b12 scaffold protein
2BOD 2005-10-05 1.5 Catalytic domain of endo-1,4-glucanase Cel6A from Thermobifida fusca in complex with methyl cellobiosyl-4-thio-beta-cellobioside
3CUI 2009-04-21 1.5 Cellulomonas fimi Xylanase/Cellulase Cex (Cf Xyn10A) in complex with sulfur substituted beta-1,4 xylotetraose
2BOF 2005-10-05 1.64 Catalytic domain of endo-1,4-glucanase Cel6A mutant Y73S from Thermobifida fusca in complex with cellotetrose
3CUF 2009-04-21 1.67 Cellulomonas fimi Xylanase/Cellulase Cex (Cf Xyn10A) in complex with cellobiose-like isofagomine
3CUG 2009-04-21 1.68 Cellulomonas fimi Xylanase/Cellulase Cex (Cf Xyn10A) in complex with cellotetraose-like isofagomine
3CUJ 2009-04-21 1.7 Cellulomonas fimi Xylanase/Cellulase Cex (Cf Xyn10A) in complex with sulfur substituted beta-1,4 xylopentaose.
1FH9 2000-08-23 1.72 CRYSTAL STRUCTURE OF THE XYLANASE CEX WITH XYLOBIOSE-DERIVED LACTAM OXIME INHIBITOR
1TML 1994-01-31 1.8 CRYSTAL STRUCTURE OF THE CATALYTIC DOMAIN OF A THERMOPHILIC ENDOCELLULASE
2EXO 1995-02-07 1.8 CRYSTAL STRUCTURE OF THE CATALYTIC DOMAIN OF THE BETA-1,4-GLYCANASE CEX FROM CELLULOMONAS FIMI
1EXP 1997-01-27 1.8 BETA-1,4-GLYCANASE CEX-CD
1FH7 2000-08-23 1.82 CRYSTAL STRUCTURE OF THE XYLANASE CEX WITH XYLOBIOSE-DERIVED INHIBITOR DEOXYNOJIRIMYCIN
2HIS 1998-10-14 1.84 CELLULOMONAS FIMI XYLANASE/CELLULASE DOUBLE MUTANT E127A/H205N WITH COVALENT CELLOBIOSE
3CUH 2009-04-21 1.89 Cellulomonas fimi Xylanase/Cellulase Cex (Cf Xyn10A) in complex with cellotriose-like isofagomine
1FHD 2000-08-23 1.9 CRYSTAL STRUCTURE OF THE XYLANASE CEX WITH XYLOBIOSE-DERIVED IMIDAZOLE INHIBITOR
2XYL 1998-03-18 1.9 CELLULOMONAS FIMI XYLANASE/CELLULASE COMPLEXED WITH 2-DEOXY-2-FLUORO-XYLOBIOSE
1FH8 2000-08-23 1.95 CRYSTAL STRUCTURE OF THE XYLANASE CEX WITH XYLOBIOSE-DERIVED ISOFAGOMINE INHIBITOR
1J01 2002-11-06 2.0 Crystal Structure Of The Xylanase Cex With Xylobiose-Derived Inhibitor Isofagomine lactam

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Exoglucanase/xylanase P07986 GUX_CELFI
100.0 Endoglucanase E-2 P26222 GUN2_THEFU
100.0 Endoglucanase A P07984 GUNA_CELFI