Urea-induced unfolding and conformational stability of 3-isopropylmalate dehydrogenase from the Thermophile thermus thermophilus and its mesophilic counterpart from Escherichia coli.


Abstract

To reveal the basis of the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli has been studied. The urea-induced equilibrium unfolding of T. thermophilus and E. coli IPMDHs at 27 degreesC was monitored by measuring the changes in far-UV CD, intrinsic fluorescence, anilinonaphthalenesulfonic acid (ANS) binding, and catalytic activity in the presence of nonionic detergent Tween 20. For both enzymes, the spectral methods revealed a biphasic unfolding transition. The first transition was protein concentration-independent, whereas the second was protein concentration-dependent for both enzymes. The observation suggested a three-state unfolding mechanism with a dimeric intermediate. However, the intermediates of the E. coli and the T. thermophilus IPMDHs seemed to be different from each other. The intermediate of the E. coli IPMDH lost its secondary and tertiary structure more than that of the thermophilic enzyme. E. coli IPMDH lost enzymatic activity through the transition from the native to the intermediate state, though the intermediate of the T. thermophilus enzyme was still active. The unfolding process of E. coli IPMDH can be explained by a sequential unfolding of individual folding domains, while there is only a small structural perturbation in the intermediate of T. thermophilus IPMDH. The higher thermal stability of T. thermophilus IPMDH can be attributed to the increase in the extent of interaction inside the first domain which unfolded prior to the unfolding of the whole molecular structure in E. coli IPMDH. Study holds ProTherm entries: 5731, 5732, 5733, 5734 Extra Details: the transition is from native to intermediate

Submission Details

ID: ToYM75QC4

Submitter: Connie Wang

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

Version: 1

Publication Details
Motono C;Yamagishi A;Oshima T,Biochemistry (1999) Urea-induced unfolding and conformational stability of 3-isopropylmalate dehydrogenase from the Thermophile thermus thermophilus and its mesophilic counterpart from Escherichia coli. PMID:9930995
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
1CNZ 1999-06-01 1.76 3-ISOPROPYLMALATE DEHYDROGENASE (IPMDH) FROM SALMONELLA TYPHIMURIUM
2Y3Z 2011-01-19 1.83 Structure of Isopropylmalate dehydrogenase from Thermus thermophilus - apo enzyme
4F7I 2012-06-13 2.0 Structure of Isopropylmalate dehydrogenase from Thermus thermophilus in complex with IPM, Mn and NADH
4WUO 2014-11-12 2.05 Structure of the E270A Mutant Isopropylmalate dehydrogenase from Thermus thermophilus in complex with IPM, Mn and NADH
1CM7 1999-07-01 2.06 3-ISOPROPYLMALATE DEHYDROGENASE FROM ESCHERICHIA COLI
1XAC 1996-04-03 2.1 CHIMERA ISOPROPYLMALATE DEHYDROGENASE BETWEEN BACILLUS SUBTILIS (M) AND THERMUS THERMOPHILUS (T) FROM N-TERMINAL: 20% T MIDDLE 20% M RESIDUAL 60% T, MUTATED AT S82R. LOW TEMPERATURE (100K) STRUCTURE.
1G2U 2000-11-01 2.1 THE STRUCTURE OF THE MUTANT, A172V, OF 3-ISOPROPYLMALATE DEHYDROGENASE FROM THERMUS THERMOPHILUS HB8 : ITS THERMOSTABILITY AND STRUCTURE.
1XAD 1996-04-03 2.1 CHIMERA ISOPROPYLMALATE DEHYDROGENASE BETWEEN BACILLUS SUBTILIS (M) AND THERMUS THERMOPHILUS (T) FROM N-TERMINAL: 20% T MIDDLE 20% M RESIDUAL 60% T, MUTATED AT S82R. LOW TEMPERATURE (150K) STRUCTURE.
1XAA 1996-04-03 2.1 3-ISOPROPYLMALATE DEHYDROGENASE, LOW TEMPERATURE (100K) STRUCTURE
1XAB 1996-04-03 2.1 3-ISOPROPYLMALATE DEHYDROGENASE, LOW TEMPERATURE (150K) STRUCTURE
1DR0 2000-01-19 2.2 STRUCTURE OF MODIFIED 3-ISOPROPYLMALATE DEHYDROGENASE AT THE C-TERMINUS, HD708
1IDM 1995-09-15 2.2 3-ISOPROPYLMALATE DEHYDROGENASE, LOOP-DELETED CHIMERA
1IPD 1993-10-31 2.2 THREE-DIMENSIONAL STRUCTURE OF A HIGHLY THERMOSTABLE ENZYME, 3-ISOPROPYLMALATE DEHYDROGENASE OF THERMUS THERMOPHILUS AT 2.2 ANGSTROMS RESOLUTION
2Y41 2011-01-19 2.2 Structure of Isopropylmalate dehydrogenase from Thermus thermophilus - complex with IPM and MN
1WAL 1999-05-25 2.27 3-ISOPROPYLMALATE DEHYDROGENASE (IPMDH) MUTANT (M219A)FROM THERMUS THERMOPHILUS
1GC9 2000-09-27 2.3 THE CRYSTAL STRUCTURE OF THERMUS THERMOPHILUS 3-ISOPROPYLMALATE DEHYDROGENASE MUTATED AT 172TH FROM ALA TO GLY
1OSJ 1997-01-27 2.35 STRUCTURE OF 3-ISOPROPYLMALATE DEHYDROGENASE
2Y40 2011-01-19 2.5 Structure of Isopropylmalate dehydrogenase from Thermus thermophilus - complex with Mn
1GC8 2000-09-27 2.5 THE CRYSTAL STRUCTURE OF THERMUS THERMOPHILUS 3-ISOPROPYLMALATE DEHYDROGENASE MUTATED AT 172TH FROM ALA TO PHE
1HEX 1994-12-20 2.5 STRUCTURE OF 3-ISOPROPYLMALATE DEHYDROGENASE IN COMPLEX WITH NAD+: LIGAND-INDUCED LOOP-CLOSING AND MECHANISM FOR COFACTOR SPECIFICITY
2Y42 2011-01-19 2.5 Structure of Isopropylmalate dehydrogenase from Thermus thermophilus - complex with NADH and Mn
1DR8 2000-01-19 2.7 STRUCTURE OF MODIFIED 3-ISOPROPYLMALATE DEHYDROGENASE AT THE C-TERMINUS, HD177
2ZTW 2009-10-13 2.79 Structure of 3-isopropylmalate dehydrogenase in complex with the inhibitor and NAD+
1DPZ 2000-01-12 2.8 STRUCTURE OF MODIFIED 3-ISOPROPYLMALATE DEHYDROGENASE AT THE C-TERMINUS, HD711
1OSI 1997-01-27 3.0 STRUCTURE OF 3-ISOPROPYLMALATE DEHYDROGENASE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
93.7 3-isopropylmalate dehydrogenase Q8Z9I1 LEU3_SALTI
94.2 3-isopropylmalate dehydrogenase Q5PDG2 LEU3_SALPA
94.5 3-isopropylmalate dehydrogenase P37412 LEU3_SALTY
94.5 3-isopropylmalate dehydrogenase Q57TE7 LEU3_SALCH
99.2 3-isopropylmalate dehydrogenase Q32K21 LEU3_SHIDS
99.2 3-isopropylmalate dehydrogenase Q83SP1 LEU3_SHIFL
99.4 3-isopropylmalate dehydrogenase Q8FL76 LEU3_ECOL6
99.4 3-isopropylmalate dehydrogenase Q3Z5T7 LEU3_SHISS
99.4 3-isopropylmalate dehydrogenase Q8X9Z9 LEU3_ECO57
99.7 3-isopropylmalate dehydrogenase Q1RGC4 LEU3_ECOUT
99.7 3-isopropylmalate dehydrogenase Q326G2 LEU3_SHIBS
100.0 3-isopropylmalate dehydrogenase P30125 LEU3_ECOLI
91.9 3-isopropylmalate dehydrogenase P24098 LEU3_THEAQ
100.0 3-isopropylmalate dehydrogenase P61495 LEU3_THETH
99.4 3-isopropylmalate dehydrogenase P61494 LEU3_THET2
100.0 3-isopropylmalate dehydrogenase Q5SIY4 LEU3_THET8