Chemistry Weekly Seminar - PhD Candidate Natasha Vetter

Title:

Using reaction kinetics and synthetic substrate analogues to study the unusual enzyme glucose-6-phosphate 3-dehydrogenase

Abstract:

Kanosamine is an amino sugar produced by a wide variety of bacteria, notably Bacillus species. This sugar is a natural antibiotic and fungicide, and is found in many other more complex antibiotics, such as kanamycin, tobramycin, and rifamycins., We discovered a novel kanosamine biosynthetic pathway in Bacillus subtilis, one that begins with the oxidation of glucose 6-phosphate (G6P) by NtdC, an NAD-dependent G6P 3-dehydrogenase. The product, 3-oxo-d-glucose 6-phosphate (3oG6P), has never been synthesized or isolated, and under alkaline conditions, is not stable due to 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.

In this talk, I will discuss the characterization NtdC as well as the synthesis of a variety of substrate analogues. We have developed two methods to assay NtdC and determine the kinetic parameters with its natural substrate, G6P. Our first method involves the deconvolution of the evolving UV spectrum, which has allowed us to simultaneously measure the appearance of both products. This has allowed us to characterize both the enzymatic reaction and non-enzymatic enolate formation. Our second method involves coupling NtdC with NtdA, which consumes 3oG6P as it is formed, allowing direct measurement of NADH formation. The second part of the talk will focus on the synthesis of substrate analogues designed to give a more complete understanding of the NtdC reaction, to provide insight into important features for substrate binding, and to assess stereo- and regiochemical preferences of the enzyme.