Trans-N-deoxyribosylase: purification by affinity chromatography and characterization

Area of Science:

• Biochemistry
• Enzymology
• Microbial genetics

Background:

• Trans-N-deoxyribosylase (EC 2.4.2.6) is traditionally viewed as a single enzyme.
• This enzyme is known to catalyze the transfer of deoxyribosyl moieties to and from both purine and pyrimidine bases.

Purpose of the Study:

• To investigate the potential for distinct trans-N-deoxyribosylase activities within Lactobacillus helveticus.
• To separate and characterize these enzymes if they exist.

Main Methods:

• Affinity chromatography using two types of ligands was employed to purify enzymes from Lactobacillus helveticus extract.
• Enzyme activity was assessed for substrate specificity (purine vs. pyrimidine bases).
• Tris inhibition studies were conducted to compare enzyme susceptibility.

Main Results:

• Two distinct trans-N-deoxyribosylases were successfully separated and purified.
• Trans-N-deoxyribosylase-I was identified as purine-specific.
• Trans-N-deoxyribosylase-II demonstrated activity with both purine and pyrimidine bases.
• The two enzymes exhibited different susceptibility to Tris inhibition.
• Preliminary data suggested trans-N-deoxyribosylase-I is a polymeric enzyme with a molecular weight of approximately 86,000 Da.

Conclusions:

• Lactobacillus helveticus possesses at least two distinct trans-N-deoxyribosylases with differing substrate specificities.
• These enzymes, trans-N-deoxyribosylase-I and trans-N-deoxyribosylase-II, exhibit unique biochemical properties.
• Further characterization of these enzymes could reveal novel insights into deoxyribosyl transfer mechanisms.