Thermodynamic basis of the thermostability of CYP175A1 from Thermus thermophilus.


Abstract

Detailed circular dichroism studies have been carried out to monitor thermal as well as denaturant induced unfolding of CYP175A1 from Thermus thermophilus and its mesophilic homologue, CYP101 from Pseudomonas putida. The unfolding midpoint temperatures for tertiary and secondary structures of the substrate-free CYP175A1 were found to be 83.7 degrees C and 87 degrees C respectively, while the corresponding midpoint temperatures for substrate-free CYP101 were at 47.7 degrees C and 51 degrees C respectively. The apparent C(m) value for GdnHCl induced unfolding of secondary structure of CYP175A1 was found to be 2.6M. The thermodynamic stability curves determined from GdnHCl induced unfolding of the enzymes at different temperatures, showed that CYP175A1 had higher free energy compared to CYP101 at temperature >10 degrees C. The results clearly established that the high thermostability of CYP175A1 arises predominantly due to higher enthalpy of the thermostable enzyme compared to CYP101. The larger number of salt bridges was proposed to be responsible for higher enthalpy of stabilization of CYP175A1. Study holds ProTherm entries: 25835, 25836, 25837, 25838, 25839, 25840, 25841, 25842, 25843, 25844 Extra Details: Thermodynamic parameter for secondary structure of CYP175A1 Cytochrome P450; CYP175A1; Circular dichroism; Stability curve; Thermostability; Enthalpy

Submission Details

ID: kwX9pcUH4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:56 p.m.

Version: 1

Publication Details
Behera RK;Mazumdar S,Int. J. Biol. Macromol. (2010) Thermodynamic basis of the thermostability of CYP175A1 from Thermus thermophilus. PMID:20138909
Additional Information

Study Summary

Number of data points 21
Proteins Cytochrome P450 (Cyp175a1) ; Camphor 5-monooxygenase ; Camphor 5-monooxygenase ; Cytochrome P450
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: dCp temp:14.0 C ; Experimental Assay: dHvH temp:14.0 C ; Experimental Assay: dG temp:14.0 C ; Experimental Assay: dCp temp:14.5 C ; Experimental Assay: dHvH temp:14.5 C ; Experimental Assay: dG temp:14.5 C ; Experimental Assay: dCp temp:35.0 C ; Experimental Assay: dHvH temp:35.0 C ; Experimental Assay: dG temp:35.0 C ; Experimental Assay: dCp temp:29.0 C ; Experimental Assay: dHvH temp:29.0 C ; Experimental Assay: dG temp:29.0 C ; Experimental Assay: Tm
Libraries Mutations for sequence TTETIQSNANLAPLPPHVPEHLVFDFDMYNPSNLSAGVQEAWAVLQESNVPDLVWTRCNGGHWIATRGQLIREAYEDYRHFSSECPFIPREAGEAYDFIPTSMDPPEQRQFRALANQVVGMPVVDKLENRIQELACSLIESLRPQGQCNFTEDYAEPFPIRIFMLLAGLPEEDIPHLKYLTDQMTRPDGSMTFAEAKEALYDYLIPIIEQRRQKPGTDAISIVANGQVNGRPITSDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELIQRPERIPAACEELLRRFSLVADGRILTSDYEFHGVQLKKGDQILLPQMLSGLDERENACPMHVDFSRQKVSHTTFGHGSHLCLGQHLARREIIVTLKEWLTRIPDFSIAPGAQIQHKSGIVSGVQALPLVWDPATTKAV ; Mutations for sequence MKRLSLREAWPYLKDLQQDPLAVLLEWGRAHPRLFLPLPRFPLALIFDPEGVEGALLAEGTTKATFQYRALSRLTGRGLLTDWGKSWKEARKALKDPFLPKSVRGYREAMEEEAWAFFGEWRGEERDLDHEMLALSLRLLGRALFGKPLSPSLAEHALKALDRIMAQTRSPLALLDLAAEARFRKDRGALYREAEALIVHPPLSHLPRERALSEAVTLLVAGHETVASALTWSFLLLSHRPDWQKRVAESEEAALAAFQEALRLYPPAWILTRRLERPLLLGEDRLPQGTTLVLSPYVTQRLYFPEGEAFQPERFLAERGTPSGRYFPFGLGQRLCLGRDFALLEGPIVLRAFFRRFRLDPLPFPRVLAQVTLRPEGGLPARPREGVRA

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
3WRK 2014-02-25T00:00:00+0000 2.61 Crystal structure of P450cam
3WRM 2014-02-25T00:00:00+0000 1.95 Crystal structure of P450cam
2CP4 1991-06-04T00:00:00+0000 2.1 CRYSTAL STRUCTURE OF THE CYTOCHROME P450-CAM ACTIVE SITE MUTANT THR252ALA
4JWU 2013-03-27T00:00:00+0000 2.2 Crystal structure of Cytochrome P450cam-putidaredoxin complex
3WRH 2014-02-25T00:00:00+0000 1.62 Crystal structure of P450cam
1PHE 1992-07-27T00:00:00+0000 1.6 CRYSTAL STRUCTURES OF METYRAPONE-AND PHENYLIMIDAZOLE-INHIBITED COMPLEXES OF CYTOCHROME P450-CAM
4KKY 2013-05-06T00:00:00+0000 2.0 Crystal structure of N-(1-Pyrene)acetamide labeled P450cam in substrate bound form.
3P6R 2010-10-11T00:00:00+0000 2.1 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog 3OH-AdaC1-Etg-Boc
3P6U 2010-10-11T00:00:00+0000 1.7 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC3-C6-Dans
3P6O 2010-10-11T00:00:00+0000 2.0 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC1-Etg-Dans

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Camphor 5-monooxygenase P00183 CPXA_PSEPU