NDLI: Conversion of an Engineered Potassium-binding Site into a Calcium-selective Site in Cytochrome c Peroxidase

Content Provider	Journal of Biological Chemistry (JBC)
Author	Bonagura, Christopher A. Bhaskar, B. Sundaramoorthy, M. Poulos, Thomas L.
Abstract	We have previously shown that the K+ site found in ascorbate peroxidase can be successfully engineered into the closely homologous peroxidase, cytochrome c peroxidase (CCP) (Bonagura, C. A., Sundaramoorthy, M., Pappa, H. S., Patterson, W. R., and Poulos, T. L. (1996) Biochemistry 35, 6107–6115; Bonagura, C. A., Sundaramoorthy, M., Bhaskar, B., and Poulos, T. L. (1999) Biochemistry 38, 5538–5545). All other peroxidases bind Ca2+ rather than K+. Using the K+-binding CCP mutant (CCPK2) as a template protein, together with observations from structural modeling, mutants were designed that should bind Ca2+ selectively. The crystal structure of the first generation mutant, CCPCA1, showed that a smaller cation, perhaps Na+, is bound instead of Ca2+. This is probably because the full eight-ligand coordination sphere did not form owing to a local disordering of one of the essential cation ligands. Based on these observations, a second mutant, CCPCA2, was designed. The crystal structure showed Ca2+ binding in the CCPCA2 mutant and a well ordered cation-binding loop with the full complement of eight protein to cation ligands. Because cation binding to the engineered loop results in diminished CCP activity and destabilization of the essential Trp191 radical as measured by EPR spectroscopy, these measurements can be used as sensitive methods for determining cation-binding selectivity. Both activity and EPR titration studies show that CCPCA2 binds Ca2+ more effectively than K+, demonstrating that an iterative protein engineering-based approach is important in switching protein cation selectivity.
Related Links	http://www.jbc.org/content/274/53/37827.abstract
Ending Page	37833
Starting Page	37827
Page Count	7
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	53
Volume Number	274
DOI	10.1074/jbc.274.53.37827
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1999-12-31
Access Restriction	Open
Subject Keyword	Cytochromec peroxidase (CCP) 4-morpholineethanesulfonic acid (MES) PROTEIN CHEMISTRY AND STRUCTURE
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

The Structure of an Electron Transfer Complex Containing a Cytochrome c and a Peroxidase

Preserved Catalytic Activity in an Engineered Ribonucleotide Reductase R2 Protein with a Nonphysiological Radical Transfer Pathway THE IMPORTANCE OF HYDROGEN BOND CONNECTIONS BETWEEN THE PARTICIPATING RESIDUES

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase III. PREDICTION OF THE DOCKED COMPLEX BY A COMPLETE, SYSTEMATIC SEARCH

Structure and Characterization of Ectothiorhodospira vacuolata Cytochrome b 558, a Prokaryotic Homologue of Cytochrome b 5

Identification of the Calcium Binding Site and a Novel Ytterbium Site in Blood Coagulation Factor XIII by X-ray Crystallography

Dissociation and Aggregation of Calpain in the Presence of Calcium

Flux Control of Cytochrome c Oxidase in Human Skeletal Muscle

A Cooperative Oxygen Binding Hemoglobin from Mycobacterium tuberculosis STABILIZATION OF HEME LIGANDS BY A DISTAL TYROSINE RESIDUE

Loss of Either of the Two Heme-binding Cysteines from a Class I c-Type Cytochrome Has a Surprisingly Small Effect on Physicochemical Properties

Conversion of an Engineered Potassium-binding Site into a Calcium-selective Site in Cytochrome c Peroxidase

Similar Documents

The Structure of an Electron Transfer Complex Containing a Cytochrome c and a Peroxidase

Preserved Catalytic Activity in an Engineered Ribonucleotide Reductase R2 Protein with a Nonphysiological Radical Transfer Pathway THE IMPORTANCE OF HYDROGEN BOND CONNECTIONS BETWEEN THE PARTICIPATING RESIDUES

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase III. PREDICTION OF THE DOCKED COMPLEX BY A COMPLETE, SYSTEMATIC SEARCH

Structure and Characterization of Ectothiorhodospira vacuolata Cytochrome b 558, a Prokaryotic Homologue of Cytochrome b 5

Identification of the Calcium Binding Site and a Novel Ytterbium Site in Blood Coagulation Factor XIII by X-ray Crystallography

Dissociation and Aggregation of Calpain in the Presence of Calcium

Flux Control of Cytochrome c Oxidase in Human Skeletal Muscle

A Cooperative Oxygen Binding Hemoglobin from Mycobacterium tuberculosis STABILIZATION OF HEME LIGANDS BY A DISTAL TYROSINE RESIDUE

Loss of Either of the Two Heme-binding Cysteines from a Class I c-Type Cytochrome Has a Surprisingly Small Effect on Physicochemical Properties

Conversion of an Engineered Potassium-binding Site into a Calcium-selective Site in Cytochrome c Peroxidase