A novel selective nucleoside phosphorylating enzyme from Morganella morganii

Area of Science:

• Biochemistry
• Enzymology
• Microbiology

Background:

• Nucleoside phosphorylating enzymes are crucial for nucleotide metabolism.
• Understanding their properties can lead to novel biotechnological applications.
• Morganella morganii is a bacterial species with diverse metabolic capabilities.

Purpose of the Study:

• To purify and characterize a selective nucleoside phosphorylating enzyme from Morganella morganii.
• To investigate the enzyme's substrate specificity, kinetic properties, and potential as an acid phosphatase.
• To explore the enzyme's utility in the synthesis of nucleoside-5 omino-monophosphates.

Main Methods:

• Purification of the enzyme to homogeneity from crude extract.
• Determination of molecular mass and subunit composition.
• Enzyme assays for phosphorylating and phosphatase activities using various substrates and phosphate donors.
• Kinetic studies to determine enzyme characteristics.
• Synthesis of inosine-5 omino-monophosphate (5 omino-IMP) using the purified enzyme.

Main Results:

• A six-subunit enzyme (M(r) 25,000 per subunit) was purified.
• The enzyme selectively phosphorylated nucleosides at the 5 omino-position using pyrophosphate, carbamylphosphate, or acetylphosphate.
• It exhibited acid phosphatase activity, dephosphorylating phosphate esters but weakly affecting nucleoside-3 omino-monophosphates.
• Enzyme activity was inhibited by sulfhydryl reagents and heavy metals but not EDTA.
• Synthesis of 32.6 mM 5 omino-IMP with a 41% molar yield was achieved, though subsequent hydrolysis occurred.

Conclusions:

• The purified enzyme is a selective nucleoside phosphorylating agent with acid phosphatase characteristics.
• The enzyme demonstrates potential for the synthesis of specific nucleoside-5 omino-monophosphates.
• Further optimization is needed to prevent product hydrolysis during synthesis.