Role of threonine 101 on the stability of the heme active site of cytochrome P450cam: multiwavelength circular dichroism studies.


Abstract

The role of the threonine 101 residue that resides close to the heme propionic acid side chain of cytochrome P450cam on the conformational properties of the active site of the enzyme has been investigated by circular dichroism (CD) spectroscopy. Site-specific mutation of the threonine by valine has been carried out that does not affect the size of the residue but significantly alters the hydropathy index. The T101V mutant of cytochrome P450cam showed distinct differences in the CD spectra near the heme region, indicating a subtle effect of the mutation on the properties of the heme active site. Thermal stabilities of the mutant and wild-type enzyme have been studied by temperature dependence of the ellipticity (intensity of the CD band) in the far-UV region for the secondary structure and at different wavelengths in the visible region that arise from the heme moiety for the tertiary structure around the prosthetic group. The thermal unfolding data from variations of the CD intensity at different wavelengths were analyzed using a generalized multistep unfolding model, and two distinct equilibrium intermediate conformational states of the enzyme were identified. The mutation of the T101 residue by valine was found to decrease the thermal stability of both the intermediates in the presence of the substrate. On the other hand, this mutation had no apparent effect on the thermal stability of the enzyme in the absence of the substrate. These results suggested that the threonine residue stabilizes the protein cavity around the heme center in the case of the substrate-bound species, possibly by hydrogen bonding with one of the propionate side chains of the heme moiety. Such hydrogen bonding of the heme propionate with threonine is absent in the substrate-free form of the enzyme. Study holds ProTherm entries: 20252, 20253, 20254, 20255, 20256, 20257, 20258, 20259, 20260, 20261, 20262, 20263, 20264, 20265 Extra Details: Far-UV CD was monitored at 221 nm in the presence of 1 mM (IR)-Camphor. conformational properties, heme region, thermal stability

Submission Details

ID: sfSBSotf

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
Manna SK;Mazumdar S,Biochemistry (2006) Role of threonine 101 on the stability of the heme active site of cytochrome P450cam: multiwavelength circular dichroism studies. PMID:17042489
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
2M56 2013-08-21 The structure of the complex of cytochrome P450cam and its electron donor putidaredoxin determined by paramagnetic NMR spectroscopy
2L8M 2011-02-16 Reduced and CO-bound cytochrome P450cam (CYP101A1)
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)
2FE6 2006-03-14 1.5 P450CAM from Pseudomonas putida reconstituted with manganic protoporphyrin IX
2H7Q 2006-10-31 1.5 Cytochrome P450cam complexed with imidazole
3L61 2010-04-21 1.5 Crystal structure of substrate-free P450cam at 200 mM [K+]
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)
5IK1 2016-05-04 1.53 Open state of P450cam after soaking in camphor
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
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
3FWG 2009-03-03 1.55 Ferric camphor bound Cytochrome P450cam, Arg365Leu, Glu366Gln, monoclinic crystal form
1QMQ 2000-10-06 1.55 Optical detection of cytochrome P450 by sensitizer-linked substrates
2ZUJ 2009-10-20 1.6 Crystal Structure of Camphor-soaked Ferric Cytochrome P450cam Mutant(D297L)
1PHB 1993-10-31 1.6 INHIBITOR-INDUCED CONFORMATIONAL CHANGE IN CYTOCHROME P450-CAM
1PHE 1993-10-31 1.6 CRYSTAL STRUCTURES OF METYRAPONE-AND PHENYLIMIDAZOLE-INHIBITED COMPLEXES OF CYTOCHROME P450-CAM
1PHD 1993-10-31 1.6 CRYSTAL STRUCTURES OF METYRAPONE-AND PHENYLIMIDAZOLE-INHIBITED COMPLEXES OF CYTOCHROME P450-CAM
1PHF 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
1PHG 1993-10-31 1.6 CRYSTAL STRUCTURES OF METYRAPONE-AND PHENYLIMIDAZOLE-INHIBITED COMPLEXES OF CYTOCHROME P450-CAM
1DZ4 2000-07-20 1.6 ferric p450cam from pseudomonas putida
2ZAX 2008-01-01 1.6 Crystal Structure of Ferric Cytochrome P450cam
3WRH 2015-03-18 1.62 Crystal structure of P450cam
2CPP 1987-07-16 1.63 HIGH-RESOLUTION CRYSTAL STRUCTURE OF CYTOCHROME P450-CAM
1PHA 1993-10-31 1.63 INHIBITOR-INDUCED CONFORMATIONAL CHANGE IN CYTOCHROME P450-CAM
3OIA 2010-11-17 1.65 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC1-C8GluEtg-Bio
1K2O 2001-10-17 1.65 Cytochrome P450Cam with Bound BIS(2,2'-BIPYRIDINE)-(5-METHYL-2-2'-BIPYRIDINE)-C2-ADAMANTANE RUTHENIUM (II)
3WRL 2015-03-18 1.65 Crystal structure of P450cam
3P6X 2010-11-17 1.65 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC3-C8-Dans
3P6N 2010-11-17 1.7 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC1-C8-Dans
1YRD 2005-02-15 1.7 X-ray crystal structure of PERDEUTERATED Cytochrome P450cam
2FEU 2006-03-14 1.7 P450CAM from Pseudomonas putida reconstituted with manganic protoporphyrin IX
5GXG 2017-01-18 1.7 High-resolution crystal structure of the electron transfer complex of cytochrome p450cam with putidaredoxin
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
3P6U 2010-11-17 1.7 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC3-C6-Dans
3L62 2010-04-21 1.7 Crystal structure of substrate-free P450cam at low [K+]
3OL5 2010-11-17 1.75 Crystal Structure of Cytochrome P450cam crystallized with a tethered substrate analog 3OH-AdaC1-C8-Dans
5CP4 1998-09-16 1.75 CRYOGENIC STRUCTURE OF P450CAM
2QBN 2007-12-25 1.75 Crystal structure of ferric G248V cytochrome P450cam
2QBM 2007-12-25 1.8 Crystal structure of the P450cam G248T mutant in the cyanide bound state
1T87 2004-05-25 1.8 Crystal Structure of the Ferrous CO-bound Cytochrome P450cam (C334A)
1T85 2004-06-01 1.8 Crystal Structure of the Ferrous CO-bound Cytochrome P450cam Mutant (L358P/C334A)
1AKD 1997-11-19 1.8 CYTOCHROME P450CAM FROM PSEUDOMONAS PUTIDA, COMPLEXED WITH 1S-CAMPHOR
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)
1O76 2002-12-19 1.8 CYANIDE COMPLEX OF P450CAM FROM PSEUDOMONAS PUTIDA
2QBL 2007-12-25 1.8 Crystal structure of ferric G248T cytochrome P450cam
2Z97 2008-09-23 1.8 Crystal Structure of Ferric Cytochrome P450cam Reconstituted with 7-Methyl-7-depropionated Hemin
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
3CPP 1990-04-15 1.9 CRYSTAL STRUCTURE OF THE CARBON MONOXY-SUBSTRATE-CYTOCHROME P450-CAM TERNARY COMPLEX
1T86 2004-05-25 1.9 Crystal Structure of the Ferrous Cytochrome P450cam Mutant (L358P/C334A)
3FWJ 2009-03-03 1.9 Ferric camphor bound Cytochrome P450cam containing a selenocysteine as the 5th heme ligand, orthorombic crystal form
6CPP 1991-07-15 1.9 CRYSTAL STRUCTURES OF CYTOCHROME P450-CAM COMPLEXED WITH CAMPHANE, THIOCAMPHOR, AND ADAMANTANE: FACTORS CONTROLLING P450 SUBSTRATE HYDROXYLATION
2QBO 2007-12-25 1.9 Crystal structure of the P450cam G248V mutant in the cyanide bound state
6CP4 1998-09-16 1.9 P450CAM D251N MUTANT
1DZ6 2000-03-30 1.9 ferrous p450cam from pseudomonas putida
1T88 2004-05-25 1.9 Crystal Structure of the Ferrous Cytochrome P450cam (C334A)
1CP4 1993-01-15 1.9 FORMATION, CRYSTAL STRUCTURE, AND REARRANGEMENT OF A CYTOCHROME P450-CAM IRON-PHENYL COMPLEX
1DZ8 2000-03-31 1.9 oxygen complex of p450cam from pseudomonas putida
3P6P 2010-11-17 1.9 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC1-C6-Bio
2A1N 2005-07-05 1.9 Crystal structure of ferrous dioxygen complex of D251N cytochrome P450cam
1DZ9 2000-03-30 1.9 Putative oxo complex of P450cam from Pseudomonas putida
3P6T 2010-11-17 1.9 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC2-C8-Dans
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
3P6V 2010-11-17 2.0 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog 3Et-AdaC1-Etg-Boc
3P6S 2010-11-17 2.0 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC2-C8-Dans
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
3P6M 2010-11-17 2.0 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC1-C8-Dans
1GEM 2000-12-06 2.0 STRUCTURAL CHARACTERIZATION OF N-BUTYL-ISOCYANIDE COMPLEXES OF CYTOCHROMES P450NOR AND P450CAM
7CPP 1991-07-15 2.0 THE STRUCTURAL BASIS FOR SUBSTRATE-INDUCED CHANGES IN REDOX POTENTIAL AND SPIN EQUILIBRIUM IN CYTOCHROME P450(CAM)
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
1UYU 2005-03-07 2.0 Xenon COMPLEX OF wildtype P450CAM FROM PSEUDOMONAS PUTIDA
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
4KKY 2014-05-21 2.0 Crystal structure of N-(1-Pyrene)acetamide labeled P450cam in substrate bound form.
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
4L4B 2013-07-31 2.1 Structure of L358A/K178G/D182N mutant of P450cam bound to camphor
2CP4 1993-01-15 2.1 CRYSTAL STRUCTURE OF THE CYTOCHROME P450-CAM ACTIVE SITE MUTANT THR252ALA
2GQX 2007-04-17 2.1 Crystal structure of cytochrome p450cam mutant (f87w/y96f/l244a/v247l/c334a) with pentachlorobenzene
4CP4 1993-01-15 2.1 CRYSTAL STRUCTURE OF THE CYTOCHROME P450-CAM ACTIVE SITE MUTANT THR252ALA
3P6R 2010-11-17 2.1 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog 3OH-AdaC1-Etg-Boc
3P6W 2010-11-17 2.1 Crystal Structure of Cytochrome P450cam crystallized in the presence of a tethered substrate analog AdaC3-Etg-Boc
4L4A 2013-07-31 2.1 Structure of L358A/K178G mutant of P450cam bound to camphor
2H7R 2006-10-31 2.1 L244A mutant of Cytochrome P450cam complexed with imidazole
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)
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
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
6NBL 2019-03-06 2.15 Cytochrome P450cam-putidaredoxin complex bound to camphor and cyanide
2H7S 2006-10-31 2.15 L244A mutant of Cytochrome P450cam
1J51 2002-01-23 2.2 CRYSTAL STRUCTURE OF CYTOCHROME P450CAM MUTANT (F87W/Y96F/V247L/C334A) WITH 1,3,5-TRICHLOROBENZENE
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)
4L4C 2013-07-31 2.2 Structure of L358P/K178G mutant of P450cam bound to camphor
4G3R 2012-08-01 2.2 Crystal Structure of Nitrosyl Cytochrome P450cam
1NOO 1996-03-08 2.2 CYTOCHROME P450-CAM COMPLEXED WITH 5-EXO-HYDROXYCAMPHOR
4JWU 2013-06-19 2.2 Crystal structure of Cytochrome P450cam-putidaredoxin complex
1GJM 2001-07-31 2.2 Covalent attachment of an electroactive sulphydryl reagent in the active site of cytochrome P450cam
3CP4 1993-01-15 2.3 CRYSTAL STRUCTURE OF THE CYTOCHROME P450-CAM ACTIVE SITE MUTANT THR252ALA
1P2Y 2003-10-28 2.3 CRYSTAL STRUCTURE OF CYTOCHROME P450CAM IN COMPLEX WITH (S)-(-)-NICOTINE
2GR6 2007-04-17 2.3 Crystal structure of cytochrome p450cam mutant (f87w/y96f/l244a/v247l/c334a)
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