Substrate Substitution in Kanosamine Biosynthesis Using Phosphonates and Phosphite Rescue

Abstract

Kanosamine is an antibiotic and antifungal compound synthesized from glucose 6-phosphate (G6P) in Bacillus subtilis by the action of three enzymes: NtdC, which catalyzes NAD-dependent oxidation of the C3-hydroxyl; NtdA, a PLP-dependent aminotransferase; and NtdB, a phosphatase. We previously demonstrated that NtdC can also oxidize substrates such as glucose and xylose, though at much lower rates, suggesting that the phosphoryloxymethylene moiety of the substrate is critical for effective catalysis. To probe this, we synthesized two phosphonate analogues of G6P in which the bridging oxygen is replaced by methylene and difluoromethylene groups. These analogues are substrates for NtdC, with second-order rate constants an order of magnitude lower than those for G6P. NtdA converts the resulting 3-keto products to the corresponding kanosamine 6-phosphonate analogues. We compared the rates to the rate of NtdC oxidation of glucose and xylose and showed that the low reactivity of xylose could be rescued 4-fold by the presence of phosphite, mimicking G6P in two pieces. These results allow the evaluation of the individual energetic contributions to catalysis of the bridging oxygen, the bridging C6 methylene, the phosphodianion, and the entropic gain of one substrate versus two substrate pieces. Phosphite also rescued the reversible formation 3-amino-3-deoxy-d-xylose by NtdA, demonstrating that truncated and nonhydrolyzable analogues of kanosamine 6-phosphate can be generated enzymatically.

Comparative Genomics and Physiological Characterization of Two Aerobic Spore Formers Isolated from Human Ileal Samples

“…In SF106, the specific NRPS for Bacilysin production and maturation are clustered, as in other members of the B. subtilis species ( Supplementary Materials Figure S2 , panel B). Kanosamine is an amino sugar with antifungal activity synthesized in various Bacillus species by the action of three enzymes, NtdC, NtdA and NtdC [ 35 ], encoded by the ntdABC operon in B. subtilis or kabABC in B. cereus [ 36 ]. In SF106 there are five genes putatively involved in kanosamine synthesis ( Supplementary Materials Figure S2 , panel C).…”

“…Further ad hoc investigations will be needed to fully clarify this point. In addition, cell-free supernatants of both strains were active against a reference strain of Candida albicans , activity that, for SF106, could be due to the reported antifungal properties of kanosamine [ 35 ]. The results presented in Table 4 are in good agreement with the bioinformatic prediction for SF106, while for SF174, the expected activity against a wide range of gram-positives was not observed, suggesting that the identified genes coding for Gallidermin are either poorly or not expressed in the experimental conditions used.…”