Simultaneous Measurement of Glucose-6-phosphate 3-Dehydrogenase (NtdC) Catalysis and the Nonenzymatic Reaction of Its Product: Kinetics and Isotope Effects on the First Step in Kanosamine Biosynthesis

Abstract

Glucose-6-phosphate 3-dehydrogenase (NtdC) is an NAD-dependent oxidoreductase encoded in the NTD operon of Bacillus subtilis. The oxidation of glucose 6-phosphate by NtdC is the first step in kanosamine biosynthesis. The product, 3-oxo-d-glucose 6-phosphate (3oG6P), has never been synthesized or isolated. The NtdC-catalyzed reaction is very slow at low and neutral pH, and its rate increases to a maximum near pH 9.5. However, under alkaline conditions, the product is not stable because of ring opening followed by deprotonation of the 1,3-dicarbonyl compound. The absorbance band due to this enolate at 310 nm overlaps with that of the other enzymatic product, NADH, complicating kinetic measurements. We report the deconvolution of the resulting spectra of the reaction to determine the rate constants and likely kinetic mechanism. In doing so, we were able to determine the extinction coefficient of the enolate of 3oG6P (23000 M cm), which allowed the measurement of the first-order rate constant (5.51 × 10 s) and activation energy (93 kJ mol) of nonenzymatic enolate formation. Using deuterium-labeled substrates, we show that hydride transfer from carbon 3 is partially rate-limiting in the enzymatic reaction, and deuterium substitution on carbon 2 has no significant effect on the enzymatic reaction but lowers the rate of deprotonation of 3oG6P 4-fold. These experiments clearly establish the regiochemistry of the reactions. Coupling of the NtdC reaction with the subsequent step in the pathway, NtdA-catalyzed glutamate-dependent amino transfer, has a small but significant effect on the rate of NAD reduction, consistent with these enzymes working together to process the unstable metabolite.

Substrate Substitution in Kanosamine Biosynthesis Using Phosphonates and Phosphite Rescue

“…This indicates that NtdA could consume the 3-keto phosphonate products, converting them to the corresponding kanosamine phosphonates. Additionally, the apparent rate of the NtdC reaction with 1 and 2 was enhanced in the presence of NtdA, as was previously observed for the reaction with G6P, 8,15 but not to the same extent, suggesting NtdA is not as tolerant of the phosphonate analogues of 3oG6P.…”

“…Observation of the complete UV spectra evolving over time revealed two peaks corresponding to formation of both NADH (340 nm) and enolate (310 nm) (Figure 2A−C), similar to what is observed with G6P. 8,15 When the reaction mixture was supplemented with excess L-glutamate and the next enzyme in the pathway, NtdA, only NADH production was observed, while the 310 nm peak was no longer observed (Figure 2D− F). This indicates that NtdA could consume the 3-keto phosphonate products, converting them to the corresponding kanosamine phosphonates.…”

“…26,27 We have previously observed that with the natural substrate, G6P, NtdC follows a random bisubstrate mechanism, and kinetic isotope effect experiments revealed that the hydride transfer step is at least partially rate-limiting. 15 The observed decreases in the k cat values suggest that hydride transfer is fully rate-limiting for these substrates.…”

“…Kinetic parameters are summarized in Table 1 and compared to those of G6P. 15 Both 1 and 2 were shown to be good substrates for NtdC, with k cat values 4-and 12-fold lower than for G6P, respectively. The K m parameter for 2 was on the same order of magnitude compared to that of G6P, while that of 1 was 3-fold higher.…”

Bacillus subtilis, the model Gram‐positive bacterium: 20 years of annotation refinement

“…In parallel, GswA, a member of a riboswitch family long of unknown function, has been functionally identified as a result of its ability to bind guanidine (Lilley, 2017), and control expression of a guanidinium exporter, GndCD (YkkCD). A variety of quorum‐sensing systems exists in B. subtilis , with a novel one involving kanosamine, a metabolite that also acts as an antibiotic against a variety of microbes (van Straaten et al ., 2013; Tojo et al ., 2014; Vetter and Palmer, 2017). Finally, B. subtilis is able to scavenge complex molecules made by other organisms, such as the xenosiderophore schizokinen via the specific transporter SxzYZA (Podkowa et al ., 2014).…”