Substrate Substitution in Kanosamine Biosynthesis Using Phosphonates and Phosphite Rescue

Area of Science:

• Biochemistry
• Enzymology
• Synthetic Biology

Background:

• Kanosamine is an antibiotic and antifungal compound produced by *Bacillus subtilis*.
• Its synthesis involves three enzymes: NtdC, NtdA, and NtdB, acting on glucose 6-phosphate (G6P).
• Previous work showed NtdC oxidizes other sugars at lower rates, indicating the importance of the G6P structure.

Purpose of the Study:

• To investigate the role of the phosphoryloxymethylene moiety in G6P for NtdC enzyme catalysis.
• To synthesize and characterize phosphonate analogues of G6P to probe substrate recognition.
• To evaluate the energetic contributions of different substrate features to enzymatic activity.

Main Methods:

• Synthesis of two phosphonate analogues of G6P, replacing the bridging oxygen with methylene and difluoromethylene groups.
• Enzymatic assays using NtdC and NtdA with G6P analogues and other substrates.
• Kinetic analysis to determine second-order rate constants.
• Investigation of phosphite's effect on xylose oxidation and NtdA activity.

Main Results:

• The synthesized G6P analogues were substrates for NtdC, albeit with reduced catalytic efficiency compared to G6P.
• NtdA successfully converted the resulting 3-keto products into kanosamine 6-phosphonate analogues.
• Phosphite addition significantly enhanced the oxidation of xylose by NtdC and rescued NtdA activity, suggesting a two-piece substrate mimicry.

Conclusions:

• The bridging oxygen and phosphodianion of G6P are critical for efficient NtdC catalysis.
• The study provides a framework for evaluating individual energetic contributions to enzyme catalysis.
• Enzymatic generation of truncated and nonhydrolyzable kanosamine 6-phosphate analogues is feasible using phosphite.