TY - JOUR

T1 - The production and characterization of artificial heterodimers of the restriction endonuclease EcoRV

AU - Wende, W

AU - Stahl, F

AU - Pingoud, A

N1 - Funding information: We thank Dr. F.K.Winkler for generously supplying structural information. We are grateful to Drs. A. Jeltsch, U. Selent and C. Wenz for valuable discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Pi 122/12-1),the Bundesministeriumf rBildungund Forschung(Programm Biotechnologie 2000) and the Fonds der Chemischen Industrie.

PY - 1996/10

Y1 - 1996/10

N2 - A novel approach to studying the inter- and intrasubunit communication required for the activity of homodimeric proteins is described. It was developed for the restriction endonuclease EcoRV, but should also be useful for other homodimeric enzymes. Two ecorV genes encoding different EcoRV mutants are coexpressed in the same Escherichia coli cell leading to homo- and heterodimeric variants of the enzyme. The two ecorV genes carry either a 5' extension coding for the glutathione-S-transferase or a His6-tag. The EcoRV heterodimer produced in vivo is separated from the two EcoRV homodimers and purified to homogeneity by affinity chromatography. Purified EcoRV heterodimers are stable and are not subject to reassortment of the subunits. To investigate the interdependence of the two catalytic centers, EcoRV heterodimers consisting of one subunit with wild type sequence and one subunit with amino acid substitutions in the PD...(D/E)XK motif, characteristic for the active sites of many restriction endonucleases, were produced. While the homodimeric EcoRV active site mutants are catalytically inactive, the heterodimeric EcoRV variants with one active and one inactive catalytic center display a twofold reduced activity toward oligodeoxynucleotide substrates compared to the wild type, and preferentially nick supercoiled plasmid DNA. From these results we conclude that in the wild type enzyme both catalytic centers function independently of each other.

AB - A novel approach to studying the inter- and intrasubunit communication required for the activity of homodimeric proteins is described. It was developed for the restriction endonuclease EcoRV, but should also be useful for other homodimeric enzymes. Two ecorV genes encoding different EcoRV mutants are coexpressed in the same Escherichia coli cell leading to homo- and heterodimeric variants of the enzyme. The two ecorV genes carry either a 5' extension coding for the glutathione-S-transferase or a His6-tag. The EcoRV heterodimer produced in vivo is separated from the two EcoRV homodimers and purified to homogeneity by affinity chromatography. Purified EcoRV heterodimers are stable and are not subject to reassortment of the subunits. To investigate the interdependence of the two catalytic centers, EcoRV heterodimers consisting of one subunit with wild type sequence and one subunit with amino acid substitutions in the PD...(D/E)XK motif, characteristic for the active sites of many restriction endonucleases, were produced. While the homodimeric EcoRV active site mutants are catalytically inactive, the heterodimeric EcoRV variants with one active and one inactive catalytic center display a twofold reduced activity toward oligodeoxynucleotide substrates compared to the wild type, and preferentially nick supercoiled plasmid DNA. From these results we conclude that in the wild type enzyme both catalytic centers function independently of each other.

KW - Biopolymers

KW - Catalysis

KW - Chromatography, Affinity

KW - Cloning, Molecular

KW - Deoxyribonucleases, Type II Site-Specific/genetics

KW - Electrophoresis, Polyacrylamide Gel

KW - Kinetics

KW - Mutagenesis, Site-Directed

KW - Plasmids

U2 - 10.1515/bchm3.1996.377.10.625

DO - 10.1515/bchm3.1996.377.10.625

M3 - Article

C2 - 8922590

VL - 377

SP - 625

EP - 632

JO - Biological chemistry

JF - Biological chemistry

SN - 1431-6730

IS - 10

ER -