Conformational Control of UDP-Galactopyranose Mutase Inhibition

Abstract

UDP-galactopyranose mutase (Glf or UGM) catalyzes the formation of uridine 5′-diphosphate-α-D-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme is required for the production of Galf-containing glycans. UGM is absent in mammals, but members of the Corynebacterineae suborder require UGM for cell envelope biosynthesis. The need for UGM in some pathogens has prompted the search for inhibitors that could serve as antibiotic leads. Optimizing inhibitor potency, however, has been challenging. The UGM from Klebsiella pneumoniae (KpUGM), which is not required for viability, is more effectively impeded by small-molecule inhibitors than are essential UGMs from species such as Mycobacterium tuberculosis or Corynebacterium diphtheriae. Why KpUGM is more susceptible to inhibition than other orthologs is not clear. One potential source of difference is UGM ortholog conformation. We previously determined a structure of CdUGM bound to a triazolothiadiazine inhibitor in the open form, but it was unclear whether the small molecule inhibitor bound this form or to the closed form. By varying the terminal tag (CdUGM-His6 and GSG-CdUGM), we crystallized CdUGM to capture the enzyme in different conformations. These structures reveal a pocket in the active site that can be exploited to augment inhibitor affinity. Moreover, they suggest the inhibitor binds the open form of most prokaryotic UGMs but can bind the closed form of KpUGM. This model and the structures suggest strategies to optimize inhibitor potency by exploiting UGM conformational flexibility.

Steric Control of the Rate-Limiting Step of UDP-Galactopyranose Mutase

“…The structural basis for enzyme cofactor reduction is known from the structures of oxidized Aspergillus fumigatus UGM ( Af UGM) complexed with NAD­(P)­H . The structure of the E–S Michaelis complex is known for several UGMs, including Af UGM, , another eukaryotic UGM from Trypanosoma cruzi ( Tc UGM), and bacterial UGMs. − Furthermore, the flavin–galactose adduct has been trapped in crystallo , providing direct structural evidence of flavin functioning as a nucleophile . These and other structures have also revealed large conformational changes associated with reduction of the cofactor and substrate binding …”