R plasmid dihydrofolate reductase with a dimeric subunit structure.

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Dihydrofolate reductase (DHFR) is a crucial enzyme in folate metabolism, essential for DNA synthesis.
• Antibiotic resistance, particularly to trimethoprim, is a growing public health concern.
• Plasmids carrying antibiotic resistance genes, like R483, are important vectors for studying bacterial adaptation.

Purpose of the Study:

• To purify and characterize the DHFR enzyme encoded by plasmid R483 from a trimethoprim-resistant Escherichia coli strain.
• To compare the biochemical and structural properties of R483 DHFR with other known DHFR enzymes.
• To investigate the evolutionary relationship of R483 DHFR to chromosomal and other plasmid-encoded DHFRs.

Main Methods:

• Purification of DHFR using a multi-step approach including dye-ligand chromatography, gel filtration, and polyacrylamide gel electrophoresis.
• Determination of enzyme specific activity.
• Molecular weight estimation using gel filtration and Ferguson analysis.
• Subunit analysis via SDS-PAGE.
• Immunological cross-reactivity testing using specific antibodies.
• N-terminal amino acid sequencing.

Main Results:

• DHFR from plasmid R483 was purified 2,000-fold to homogeneity with a specific activity of 250 μmol/mg/min.
• The native enzyme's molecular weight was estimated at 32,000–39,000 Da, suggesting a dimeric structure with 19,000 Da subunits.
• Antibodies against R483 DHFR showed no cross-reactivity with DHFR from other sources (plasmid R67, T4 phage, E. coli, mouse leukemia cells).
• N-terminal sequencing indicated R483 DHFR is more closely related to chromosomal DHFR than to R67 DHFR.

Conclusions:

• The DHFR encoded by plasmid R483 exhibits distinct biochemical and immunological properties compared to other DHFR enzymes.
• The dimeric structure and specific activity provide insights into its catalytic function.
• The evolutionary analysis suggests a closer relationship to the host's chromosomal DHFR, highlighting potential gene transfer or evolutionary convergence mechanisms.