Báo cáo khoa học: Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis

Tracking Interactions That Stabilize the Dimer Structure of Starch Phosphorylase from Corynebacterium callunae

Authors: Richard Griessler, Alexandra Schwarz, Jan Mucha, and Bernd Nidetzky

Field: Biochemistry / Molecular Biology

Document Summary: This study investigates the stabilization of the dimer structure of starch phosphorylase (StP) from Corynebacterium callunae, focusing on the roles of specific arginine residues (Arg234 and Arg242) within the TOWER interface region. The research involved cloning, sequencing, and heterologous expression of the StP gene in Escherichia coli. Through site-directed mutagenesis, particularly replacing arginine residues with alanine, the study aimed to elucidate their contribution to the enzyme’s stability, especially in the presence of phosphate and sulfate. Key findings indicate that Arg234 and Arg242, in their unligated state, partially destabilize the enzyme’s dimer structure. The binding of phosphate appears to induce conformational changes that enhance stability, likely through an allosteric mechanism. The study also observed an unexpected preference for sulfate over phosphate stabilization in certain mutants, suggesting complex interactions at the dimer interface.

Detailed Table of Contents:

• Introduction
• Materials and Methods
• Preparation of an oligonucleotide probe for the StP gene
• Construction of expression plasmids
• Site-directed mutagenesis
• Expression of the StP gene in Escherichia coli
• Purification and characterization of recombinant StP and mutants thereof
• Cloning and sequencing of the StP gene
• Identification of dimer contact regions in StP from the structural alignment of StP with other GPs
• Expression of the wild-type and mutagenized StP genes in E. coli, and purification and characterization of recombinant enzymes
• Determination of dissociation constants of binary enzyme-oxyanion complexes
• Stability of recombinant wild-type StP and TOWER helix mutants thereof as revealed in urea and thermal denaturation experiments
• Results
• Comparison of CD spectra of wild-type StP, R234A mutant, and R242A mutant
• Elution profile of purified recombinant His-tagged StP upon analytical gel filtration
• Comparison of the StP amino acid sequence with the sequences of EcMalP and rmGP
• Table 1. Comparison of stabilities of recombinant wild-type StP and two enzyme variants in urea and thermal denaturation experiments at pH 7.0.
• Figure 5. Stabilization of wild-type StP and the R242A mutant by phosphate (A) and sulfate (B) against urea denaturation.
• Discussion
• Relationships between StP structure and oxyanion-dependent kinetic stability
• Comparison of StP with rmGP and other a-glucan phosphorylases
• Acknowledgements
• References
• Supplementary Material