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 MKRLSLREAWPYLKDLQQDPLAVLLEWGRAHPRLFLPLPRFPLALIFDPEGVEGALLAEGTTKATFQYRALSRLTGRGLLTDWGKSWKEARKALKDPFLPKSVRGYREAMEEEAWAFFGEWRGEERDLDHEMLALSLRLLGRALFGKPLSPSLAEHALKALDRIMAQTRSPLALLDLAAEARFRKDRGALYREAEALIVHPPLSHLPRERALSEAVTLLVAGHETVASALTWSFLLLSHRPDWQKRVAESEEAALAAFQEALRLYPPAWILTRRLERPLLLGEDRLPQGTTLVLSPYVTQRLYFPEGEAFQPERFLAERGTPSGRYFPFGLGQRLCLGRDFALLEGPIVLRAFFRRFRLDPLPFPRVLAQVTLRPEGGLPARPREGVRA ; Mutations for sequence TTETIQSNANLAPLPPHVPEHLVFDFDMYNPSNLSAGVQEAWAVLQESNVPDLVWTRCNGGHWIATRGQLIREAYEDYRHFSSECPFIPREAGEAYDFIPTSMDPPEQRQFRALANQVVGMPVVDKLENRIQELACSLIESLRPQGQCNFTEDYAEPFPIRIFMLLAGLPEEDIPHLKYLTDQMTRPDGSMTFAEAKEALYDYLIPIIEQRRQKPGTDAISIVANGQVNGRPITSDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELIQRPERIPAACEELLRRFSLVADGRILTSDYEFHGVQLKKGDQILLPQMLSGLDERENACPMHVDFSRQKVSHTTFGHGSHLCLGQHLARREIIVTLKEWLTRIPDFSIAPGAQIQHKSGIVSGVQALPLVWDPATTKAV

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
2L8M 2011-02-16 Reduced and CO-bound cytochrome P450cam (CYP101A1)
2M56 2013-08-21 The structure of the complex of cytochrome P450cam and its electron donor putidaredoxin determined by paramagnetic NMR spectroscopy
2LQD 2012-05-09 Reduced and CO-bound cytochrome P450cam (CYP101A1)
4L4E 2013-07-31 1.26 Structure of cyanide and camphor bound P450cam mutant L358A/K178G
4L4F 2013-07-31 1.29 Structure of cyanide and camphor bound P450cam mutant L358A/K178G/D182N
2ZWU 2009-02-17 1.3 Crystal Structure of Camphor Soaked Ferric Cytochrome P450cam
2ZWT 2009-02-17 1.35 Crystal Structure of Ferric Cytochrome P450cam
1YRC 2005-02-15 1.4 X-ray Crystal Structure of hydrogenated Cytochrome P450cam
1RE9 2004-11-16 1.45 CRYSTAL STRUCTURE OF CYTOCHROME P450-CAM WITH A FLUORESCENT PROBE D-8-AD (ADAMANTANE-1-CARBOXYLIC ACID-5-DIMETHYLAMINO-NAPHTHALENE-1-SULFONYLAMINO-OCTYL-AMIDE)
3L63 2010-04-21 1.5 Crystal structure of camphor-bound P450cam at low [K+]
2ZUI 2009-10-20 1.5 Crystal Structure of Camphor-soaked Ferric Cytochrome P450cam Mutant (D297N)
2H7Q 2006-10-31 1.5 Cytochrome P450cam complexed with imidazole
2FE6 2006-03-14 1.5 P450CAM from Pseudomonas putida reconstituted with manganic protoporphyrin IX
3L61 2010-04-21 1.5 Crystal structure of substrate-free P450cam at 200 mM [K+]
5IK1 2016-05-04 1.53 Open state of P450cam after soaking in camphor
1QMQ 2000-10-06 1.55 Optical detection of cytochrome P450 by sensitizer-linked substrates
3FWG 2009-03-03 1.55 Ferric camphor bound Cytochrome P450cam, Arg365Leu, Glu366Gln, monoclinic crystal form
2ZUH 2009-10-20 1.55 Crystal Structure of Camphor-soaked Ferric Cytochrome P450cam Mutant (D297A)
4L4G 2013-07-31 1.55 Structure of cyanide and camphor bound P450cam mutant L358P/K178G
2A1O 2005-07-05 1.55 Crystal structure of ferrous dioxygen complex of T252A cytochrome P450cam
2ZAW 2008-01-01 1.55 Crystal Structure of Ferric Cytochrome P450cam Reconstituted with 6-Methyl-6-depropionated Hemin
1PHB 1993-10-31 1.6 INHIBITOR-INDUCED CONFORMATIONAL CHANGE IN CYTOCHROME P450-CAM
1PHD 1993-10-31 1.6 CRYSTAL STRUCTURES OF METYRAPONE-AND PHENYLIMIDAZOLE-INHIBITED COMPLEXES OF CYTOCHROME P450-CAM
1PHC 1993-10-31 1.6 CRYSTAL STRUCTURE OF SUBSTRATE-FREE PSEUDOMONAS PUTIDA CYTOCHROME P450
1PHF 1993-10-31 1.6 CRYSTAL STRUCTURES OF METYRAPONE-AND PHENYLIMIDAZOLE-INHIBITED COMPLEXES OF CYTOCHROME P450-CAM
1PHE 1993-10-31 1.6 CRYSTAL STRUCTURES OF METYRAPONE-AND PHENYLIMIDAZOLE-INHIBITED COMPLEXES OF CYTOCHROME P450-CAM
1PHG 1993-10-31 1.6 CRYSTAL STRUCTURES OF METYRAPONE-AND PHENYLIMIDAZOLE-INHIBITED COMPLEXES OF CYTOCHROME P450-CAM
2ZUJ 2009-10-20 1.6 Crystal Structure of Camphor-soaked Ferric Cytochrome P450cam Mutant(D297L)
2ZAX 2008-01-01 1.6 Crystal Structure of Ferric Cytochrome P450cam
1DZ4 2000-07-20 1.6 ferric p450cam from pseudomonas putida
3WRH 2015-03-18 1.62 Crystal structure of P450cam
1PHA 1993-10-31 1.63 INHIBITOR-INDUCED CONFORMATIONAL CHANGE IN CYTOCHROME P450-CAM
2CPP 1987-07-16 1.63 HIGH-RESOLUTION CRYSTAL STRUCTURE OF CYTOCHROME P450-CAM
1K2O 2001-10-17 1.65 Cytochrome P450Cam with Bound BIS(2,2'-BIPYRIDINE)-(5-METHYL-2-2'-BIPYRIDINE)-C2-ADAMANTANE RUTHENIUM (II)
3OIA 2010-11-17 1.65 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC1-C8GluEtg-Bio
3P6X 2010-11-17 1.65 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC3-C8-Dans
3WRL 2015-03-18 1.65 Crystal structure of P450cam
2FEU 2006-03-14 1.7 P450CAM from Pseudomonas putida reconstituted with manganic protoporphyrin IX
3P6N 2010-11-17 1.7 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC1-C8-Dans
3P6U 2010-11-17 1.7 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC3-C6-Dans
2FER 2006-03-14 1.7 P450CAM from Pseudomonas putida reconstituted with manganic protoporphyrin IX
1GEK 2000-12-06 1.7 STRUCTURAL CHARACTERIZATION OF N-BUTYL-ISOCYANIDE COMPLEXES OF CYTOCHROMES P450NOR AND P450CAM
3L62 2010-04-21 1.7 Crystal structure of substrate-free P450cam at low [K+]
5GXG 2017-01-18 1.7 High-resolution crystal structure of the electron transfer complex of cytochrome p450cam with putidaredoxin
1YRD 2005-02-15 1.7 X-ray crystal structure of PERDEUTERATED Cytochrome P450cam
5CP4 1998-09-16 1.75 CRYOGENIC STRUCTURE OF P450CAM
2QBN 2007-12-25 1.75 Crystal structure of ferric G248V cytochrome P450cam
3OL5 2010-11-17 1.75 Crystal Structure of Cytochrome P450cam crystallized with a tethered substrate analog 3OH-AdaC1-C8-Dans
2Z97 2008-09-23 1.8 Crystal Structure of Ferric Cytochrome P450cam Reconstituted with 7-Methyl-7-depropionated Hemin
2QBM 2007-12-25 1.8 Crystal structure of the P450cam G248T mutant in the cyanide bound state
1T85 2004-06-01 1.8 Crystal Structure of the Ferrous CO-bound Cytochrome P450cam Mutant (L358P/C334A)
1O76 2002-12-19 1.8 CYANIDE COMPLEX OF P450CAM FROM PSEUDOMONAS PUTIDA
1T87 2004-05-25 1.8 Crystal Structure of the Ferrous CO-bound Cytochrome P450cam (C334A)
2QBL 2007-12-25 1.8 Crystal structure of ferric G248T cytochrome P450cam
1RF9 2004-11-16 1.8 Crystal structure of cytochrome P450-cam with a fluorescent probe D-4-AD (Adamantane-1-carboxylic acid-5-dimethylamino-naphthalene-1-sulfonylamino-butyl-amide)
1AKD 1997-11-19 1.8 CYTOCHROME P450CAM FROM PSEUDOMONAS PUTIDA, COMPLEXED WITH 1S-CAMPHOR
3FWF 2009-03-03 1.83 Ferric camphor bound cytochrome P450cam containing a Selenocysteine as the 5th heme ligand, monoclinic crystal form
3WRJ 2015-03-18 1.85 Crystal structure of P450cam
3P6P 2010-11-17 1.9 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC1-C6-Bio
1DZ8 2000-03-31 1.9 oxygen complex of p450cam from pseudomonas putida
1DZ9 2000-03-30 1.9 Putative oxo complex of P450cam from Pseudomonas putida
2QBO 2007-12-25 1.9 Crystal structure of the P450cam G248V mutant in the cyanide bound state
1T86 2004-05-25 1.9 Crystal Structure of the Ferrous Cytochrome P450cam Mutant (L358P/C334A)
6CPP 1991-07-15 1.9 CRYSTAL STRUCTURES OF CYTOCHROME P450-CAM COMPLEXED WITH CAMPHANE, THIOCAMPHOR, AND ADAMANTANE: FACTORS CONTROLLING P450 SUBSTRATE HYDROXYLATION
1DZ6 2000-03-30 1.9 ferrous p450cam from pseudomonas putida
1CP4 1993-01-15 1.9 FORMATION, CRYSTAL STRUCTURE, AND REARRANGEMENT OF A CYTOCHROME P450-CAM IRON-PHENYL COMPLEX
3FWJ 2009-03-03 1.9 Ferric camphor bound Cytochrome P450cam containing a selenocysteine as the 5th heme ligand, orthorombic crystal form
3P6T 2010-11-17 1.9 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC2-C8-Dans
2A1N 2005-07-05 1.9 Crystal structure of ferrous dioxygen complex of D251N cytochrome P450cam
1T88 2004-05-25 1.9 Crystal Structure of the Ferrous Cytochrome P450cam (C334A)
3CPP 1990-04-15 1.9 CRYSTAL STRUCTURE OF THE CARBON MONOXY-SUBSTRATE-CYTOCHROME P450-CAM TERNARY COMPLEX
6CP4 1998-09-16 1.9 P450CAM D251N MUTANT
3WRM 2015-03-18 1.95 Crystal structure of P450cam
3P6Q 2010-11-17 1.95 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC2-Etg-Boc
4EK1 2012-07-25 1.97 Crystal Structure of Electron-Spin Labeled Cytochrome P450cam
5WK7 2017-09-06 1.98 P450cam mutant R186A
5WK9 2017-09-06 1.98 R186AP450cam with CN and camphor
3P6M 2010-11-17 2.0 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC1-C8-Dans
1UYU 2005-03-07 2.0 Xenon COMPLEX OF wildtype P450CAM FROM PSEUDOMONAS PUTIDA
3P6S 2010-11-17 2.0 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC2-C8-Dans
1GEM 2000-12-06 2.0 STRUCTURAL CHARACTERIZATION OF N-BUTYL-ISOCYANIDE COMPLEXES OF CYTOCHROMES P450NOR AND P450CAM
3P6O 2010-11-17 2.0 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC1-Etg-Dans
1IWI 2002-06-05 2.0 Putidaredoxin-Binding Stablilizes an Active Conformer of Cytochrome P450cam in its Reduced State; Crystal Structure of Cytochrome P450cam
7CPP 1991-07-15 2.0 THE STRUCTURAL BASIS FOR SUBSTRATE-INDUCED CHANGES IN REDOX POTENTIAL AND SPIN EQUILIBRIUM IN CYTOCHROME P450(CAM)
3P6V 2010-11-17 2.0 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog 3Et-AdaC1-Etg-Boc
4KKY 2014-05-21 2.0 Crystal structure of N-(1-Pyrene)acetamide labeled P450cam in substrate bound form.
1IWJ 2002-06-05 2.0 Putidaredoxin-Binding Stablilizes an Active Conformer of Cytochrome P450cam in its Reduced State; Crystal Structure of Mutant(109K) Cytochrome P450cam
1IWK 2002-06-05 2.0 Putidaredoxin-Binding Stablilizes an Active Conformer of Cytochrome P450cam in its Reduced State; Crystal Structure of Mutant(112K) Cytochrome P450cam
1GEB 2000-11-15 2.03 X-RAY CRYSTAL STRUCTURE AND CATALYTIC PROPERTIES OF THR252ILE MUTANT OF CYTOCHROME P450CAM
5CPP 1991-07-15 2.08 THE STRUCTURAL BASIS FOR SUBSTRATE-INDUCED CHANGES IN REDOX POTENTIAL AND SPIN EQUILIBRIUM IN CYTOCHROME P-450(CAM)
4JX1 2013-06-19 2.09 Crystal structure of reduced Cytochrome P450cam-putidaredoxin complex bound to camphor and 5-exo-hydroxycamphor
2H7R 2006-10-31 2.1 L244A mutant of Cytochrome P450cam complexed with imidazole
2A1M 2005-07-05 2.1 Crystal structure of ferrous dioxygen complex of wild-type cytochrome P450cam
2FRZ 2007-01-16 2.1 Crystal Structure of Cytochrome P450cam mutant (F87W/Y96F/V247L/C334A)
4L4D 2013-07-31 2.1 Structure of cyanide and camphor bound P450cam mutant L358A
4L4A 2013-07-31 2.1 Structure of L358A/K178G mutant of P450cam bound to camphor
4CP4 1993-01-15 2.1 CRYSTAL STRUCTURE OF THE CYTOCHROME P450-CAM ACTIVE SITE MUTANT THR252ALA
2CP4 1993-01-15 2.1 CRYSTAL STRUCTURE OF THE CYTOCHROME P450-CAM ACTIVE SITE MUTANT THR252ALA
3P6W 2010-11-17 2.1 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC3-Etg-Boc
3P6R 2010-11-17 2.1 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog 3OH-AdaC1-Etg-Boc
4L4B 2013-07-31 2.1 Structure of L358A/K178G/D182N mutant of P450cam bound to camphor
8CPP 1991-07-15 2.1 CRYSTAL STRUCTURES OF CYTOCHROME P450-CAM COMPLEXED WITH CAMPHANE, THIOCAMPHOR, AND ADAMANTANE: FACTORS CONTROLLING P450 SUBSTRATE HYDROXYLATION
1C8J 2001-05-09 2.1 CRYSTAL STRUCTURE OF CYTOCHROME P450CAM MUTANT (F87W/Y96F)
2GQX 2007-04-17 2.1 Crystal structure of cytochrome p450cam mutant (f87w/y96f/l244a/v247l/c334a) with pentachlorobenzene
4CPP 1991-07-15 2.11 CRYSTAL STRUCTURES OF CYTOCHROME P450-CAM COMPLEXED WITH CAMPHANE, THIOCAMPHOR, AND ADAMANTANE: FACTORS CONTROLLING P450 SUBSTRATE HYDROXYLATION
4L49 2013-07-31 2.13 Structure of L358A mutant of P450cam bound to camphor
4JWS 2013-06-19 2.15 Crystal structure of Cytochrome P450cam-putidaredoxin complex
2H7S 2006-10-31 2.15 L244A mutant of Cytochrome P450cam
6NBL 2019-03-06 2.15 Cytochrome P450cam-putidaredoxin complex bound to camphor and cyanide
1NOO 1996-03-08 2.2 CYTOCHROME P450-CAM COMPLEXED WITH 5-EXO-HYDROXYCAMPHOR
4L4C 2013-07-31 2.2 Structure of L358P/K178G mutant of P450cam bound to camphor
1GJM 2001-07-31 2.2 Covalent attachment of an electroactive sulphydryl reagent in the active site of cytochrome P450cam
4G3R 2012-08-01 2.2 Crystal Structure of Nitrosyl Cytochrome P450cam
1LWL 2002-12-18 2.2 Crystal Structure of Cytochrome P450-cam with a Fluorescent Probe D-8-Ad (Adamantane-1-carboxylic acid-5-dimethylamino-naphthalene-1-sulfonylamino-octyl-amide)
1J51 2002-01-23 2.2 CRYSTAL STRUCTURE OF CYTOCHROME P450CAM MUTANT (F87W/Y96F/V247L/C334A) WITH 1,3,5-TRICHLOROBENZENE
4JWU 2013-06-19 2.2 Crystal structure of Cytochrome P450cam-putidaredoxin complex
2GR6 2007-04-17 2.3 Crystal structure of cytochrome p450cam mutant (f87w/y96f/l244a/v247l/c334a)
1P2Y 2003-10-28 2.3 CRYSTAL STRUCTURE OF CYTOCHROME P450CAM IN COMPLEX WITH (S)-(-)-NICOTINE
3CP4 1993-01-15 2.3 CRYSTAL STRUCTURE OF THE CYTOCHROME P450-CAM ACTIVE SITE MUTANT THR252ALA
1MPW 2002-10-09 2.34 Molecular Recognition in (+)-a-Pinene Oxidation by Cytochrome P450cam
3FWI 2009-03-03 2.4 Ferric camphor bound Cytochrome P450cam containing a selenocysteine as the 5th heme ligand, tetragonal crystal form
3W9C 2013-08-21 2.5 Crystal structure of the electron transfer complex of cytochrome p450cam with putidaredoxin
3WRK 2015-03-18 2.61 Crystal structure of P450cam
1P7R 2003-10-28 2.85 CRYSTAL STRUCTURE OF REDUCED, CO-EXPOSED COMPLEX OF CYTOCHROME P450CAM WITH (S)-(-)-NICOTINE
3WRI 2015-03-18 2.9 Crystal structure of P450cam

Relevant UniProtKB Entries

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