Structural Basis of Substrate Binding to UDP-galactopyranose Mutase: Crystal Structures in the Reduced and Oxidized State Complexed with UDP-galactopyranose and UDP

Partha, Sarathy Karunan; Straaten, K.E. Van; Sanders, D.A.R.

doi:10.1016/j.jmb.2009.10.013

Cited by 43 publications

(39 citation statements)

References 35 publications

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“…Kinetic constants for wild type and mutant AfUGMs were determined following a kinetic assay modified from Partha et al (20). The conversion of UDP-Gal p to UDP-Gal f was monitored at 262 nm using HPLC (Agilent Technologies, 1100 Infinity).…”

Section: Methodsmentioning

confidence: 99%

“…The bacterial enzyme is a flavoprotein found as a dimer, which requires the co-factor to be in the reduced state for activity (21). The structures of prokaryotic UGMs with and without bound substrate have also been reported (20–24). Examination of these structures confirmed that the active site of UGM closes around the substrate as it binds and is controlled by an invariant arginine residue that interacts with the diphosphate of the substrate (5, 20, 24).…”

Section: Introductionmentioning

confidence: 98%

“…UGM has been well characterized from a number of bacteria, including Escherichia coli, Klebsiella pneumonia, M. tuberculosis, and Deinococcus radiodurans (17–20). The bacterial enzyme is a flavoprotein found as a dimer, which requires the co-factor to be in the reduced state for activity (21).…”

Section: Introductionmentioning

confidence: 99%

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Structural Insight into the Unique Substrate Binding Mechanism and Flavin Redox State of UDP-galactopyranose Mutase from Aspergillus fumigatus

Straaten

Routier

Sanders

2012

Journal of Biological Chemistry

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Background: UDP-galactopyranose mutase (UGM) is a critical enzyme for the proper formation of the cell wall of pathogenic microbes.Results: The structure of UGM in complex with substrate reveals novel features of substrate binding.Conclusion: Oxidation and reduction of the flavin cofactor causes rearrangements in the active site that affect substrate binding.Significance: These are the first structures of UGM from a eukaryotic pathogen.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

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Structural Insight into the Unique Substrate Binding Mechanism and Flavin Redox State of UDP-galactopyranose Mutase from Aspergillus fumigatus

Straaten

Routier

Sanders

2012

Journal of Biological Chemistry

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“…Our structure of oxidized GSG-CdUGM in complex with UDP indicates the inactive enzyme can adopt a closed conformation analogous to that observed for the catalytically competent KpUGM. Other closed UGM orthologs have been crystallized with oxidized FAD either with or without bound substrates, including DrUGM 30 , EcUGM, 3 AfUGM 51 , and TcUGM 34 . These findings suggest that the energy barrier for conformational change is small, regardless of the FAD oxidation state.…”

Section: Discussionmentioning

confidence: 99%

“…Though the position of the conserved arginine in KpUGM 28 suggests the residue mainly interacts with the pyrophosphoryl group of the UDP-Gal p substrate, in the Deinococcus radiodurans (Dr) UGM, the corresponding arginine interacts with the pyrophosphoryl group and the galactopyranose residue. 30 Studies of the M. tuberculosis UGM with a non-substrate inhibitor suggest there is an allosteric binding pocket near the active site and that occupation of this pocket prevents loop closure. 32 Eukaryotic UGMs also adopt multiple conformations, and they have a second mobile flap containing an asparagine residue involved in substrate recognition.…”

Section: Introductionmentioning

confidence: 99%

Conformational Control of UDP-Galactopyranose Mutase Inhibition

et al. 2017

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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.

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