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Loop interactions during catalysis by dihydrofolate reductase from Moritella profunda.
Enas M Behiry1, Rhiannon M Evans, Jiannan Guo
1School of Chemistry, Cardiff University , Park Place, Cardiff CF10 3AT, United Kingdom.
Biochemistry
|July 12, 2014
Summary
Cold-adapted Moritella profunda dihydrofolate reductase (MpDHFR) variants show altered catalytic roles for key protein loops compared to Escherichia coli DHFR. Loop motions critical for catalysis in EcDHFR are less significant in MpDHFR.
Area of Science:
- Biochemistry
- Enzymology
- Protein Dynamics
Background:
- Dihydrofolate reductase (DHFR) is a crucial enzyme in folate metabolism, frequently utilized as a model for studying enzyme dynamics and catalysis.
- Understanding the structure-function relationship in DHFR, particularly concerning protein loop dynamics, is key to enzyme mechanism elucidation.
Purpose of the Study:
- To investigate the role of catalytically important M20 and FG loops in the function of cold-adapted DHFR from Moritella profunda (MpDHFR).
- To compare the effects of mutations in these loops on catalytic activity between MpDHFR and the well-characterized DHFR from Escherichia coli (EcDHFR).
Main Methods:
- Site-directed mutagenesis was employed to create variants of MpDHFR and EcDHFR with altered M20 and FG loops.
- Enzyme kinetics and steady-state turnover rates were measured for wild-type and mutant enzymes.
- Comparative analysis of catalytic efficiency and reaction mechanisms between MpDHFR and EcDHFR variants.
Main Results:
- Mutations in the M20 loop impacted steady-state turnover rates in EcDHFR but not in MpDHFR, indicating differential functional roles.
- Alterations in the FG loop also exhibited distinct effects on the catalytic performance of MpDHFR and EcDHFR.
- Despite a conserved catalytic cycle, loop motions crucial for EcDHFR catalysis appear less significant for MpDHFR function.
Conclusions:
- Protein loop dynamics play differential roles in the catalytic mechanisms of cold-adapted MpDHFR and mesophilic EcDHFR.
- The findings highlight the adaptation of enzyme mechanisms to environmental conditions, such as temperature, in DHFR evolution.
- This study provides insights into how protein dynamics contribute to catalysis in enzymes from extremophiles.