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

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 MKRLSLREAWPYLKDLQQDPLAVLLEWGRAHPRLFLPLPRFPLALIFDPEGVEGALLAEGTTKATFQYRALSRLTGRGLLTDWGKSWKEARKALKDPFLPKSVRGYREAMEEEAWAFFGEWRGEERDLDHEMLALSLRLLGRALFGKPLSPSLAEHALKALDRIMAQTRSPLALLDLAAEARFRKDRGALYREAEALIVHPPLSHLPRERALSEAVTLLVAGHETVASALTWSFLLLSHRPDWQKRVAESEEAALAAFQEALRLYPPAWILTRRLERPLLLGEDRLPQGTTLVLSPYVTQRLYFPEGEAFQPERFLAERGTPSGRYFPFGLGQRLCLGRDFALLEGPIVLRAFFRRFRLDPLPFPRVLAQVTLRPEGGLPARPREGVRA ; Mutations for sequence TTETIQSNANLAPLPPHVPEHLVFDFDMYNPSNLSAGVQEAWAVLQESNVPDLVWTRCNGGHWIATRGQLIREAYEDYRHFSSECPFIPREAGEAYDFIPTSMDPPEQRQFRALANQVVGMPVVDKLENRIQELACSLIESLRPQGQCNFTEDYAEPFPIRIFMLLAGLPEEDIPHLKYLTDQMTRPDGSMTFAEAKEALYDYLIPIIEQRRQKPGTDAISIVANGQVNGRPITSDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELIQRPERIPAACEELLRRFSLVADGRILTSDYEFHGVQLKKGDQILLPQMLSGLDERENACPMHVDFSRQKVSHTTFGHGSHLCLGQHLARREIIVTLKEWLTRIPDFSIAPGAQIQHKSGIVSGVQALPLVWDPATTKAV
Sequence Assay Result Units