Carbocyclic Substrate Analogues Reveal Kanosamine Biosynthesis Begins with the α-Anomer of Glucose 6-Phosphate.

NtdC is an NAD-dependent dehydrogenase that catalyzes the conversion of glucose 6-phosphate (G6P) to 3-oxo-glucose 6-phosphate (3oG6P), the first step in kanosamine biosynthesis in and other closely-related bacteria. The NtdC-catalyzed reaction is unusual because 3oG6P undergoes rapid ring opening, resulting in a 1,3-dicarbonyl compound that is inherently unstable due to enolate formation. We have reported the steady-state kinetic behavior of NtdC, but many questions remain about the nature of this reaction, including whether it is the α-anomer, β-anomer, or open-chain form that is the substrate for the enzyme. Here, we report the synthesis of carbocyclic G6P analogues by two routes, one based upon the Ferrier II rearrangement to generate the carbocycle and one based upon a Claisen rearrangement. We were able to synthesize both pseudo-anomers of carbaglucose 6-phosphate (C6P) using the Ferrier approach, and activity assays revealed that the pseudo-α-anomer is a good substrate for NtdC, while the pseudo-β-anomer and the open-chain analogue, sorbitol 6-phosphate (S6P), are not substrates. A more efficient synthesis of α-C6P was achieved using the Claisen rearrangement approach, which allowed for a thorough evaluation of the NtdC-catalyzed oxidation of α-C6P. The requirement for the α-anomer indicates that NtdC and NtdA, the subsequent enzyme in the pathway, have co-evolved to recognize the α-anomer in order to avoid mutarotation between enzymatic steps.