Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues.

Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues. Research Abstract Details 

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Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues. Abstract Text:

Jennifer M Chad,Karunan Partha Sarathy,Todd D Gruber,Eshwari Addala,Laura L Kiessling,David A R Sanders,

The flavoenzyme UDP-galactopyranose mutase (UGM) is a mediator of cell wall biosynthesis in many pathogenic microorganisms. UGM catalyzes a unique ring contraction reaction that results in the conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UDP-Galf is an essential precursor to the galactofuranose residues found in many different cell wall glycoconjugates. Due to the important consequences of UGM catalysis, structural and biochemical studies are needed to elucidate the mechanism and identify the key residues involved. Here, we report the results of site-directed mutagenesis studies on the absolutely conserved residues in the putative active site cleft. By generating variants of the UGM from Klebsiella pneumoniae, we have identified two arginine residues that play critical catalytic roles (alanine substitution abolishes detectable activity). These residues also have a profound effect on the binding of a fluorescent UDP derivative that inhibits UGM, suggesting that the Arg variants are defective in their ability to bind substrate. One of the residues, Arg280, is located in the putative active site, but, surprisingly, the structural studies conducted to date suggest that Arg174 is not. Molecular dynamics simulations indicate that closed UGM conformations can be accessed in which this residue contacts the pyrophosphoryl group of the UDP-Gal substrates. These results provide strong evidence that the mobile loop, noted in all the reported crystal structures, must move in order for UGM to bind its UDP-galactose substrate.

Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues. Publishing Authors By Initials

JM Chad,KP Sarathy,TD Gruber,E Addala,LL Kiessling,DA Sanders,

For similar heterocyclic compounds: heterocyclic compounds, 1-ring: pyrimidines: pyrimidine nucleotides: uracil nucleotides: uridine diphosphate: uridine diphosphate sugars: uridine diphosphate galactose research abstracts see: heterocyclic compounds: heterocyclic compounds, 1-ring: pyrimidines: pyrimidine nucleotides: uracil nucleotides: uridine diphosphate: uridine diphosphate sugars: uridine diphosphate galactose research

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Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Biochemistry

VOLUME: 46

Page Numbers: 6723-32

Journal Abbreviation: Biochemistry

ISSN: 0006-2960

DAY: 19

MONTH: 05

YEAR: 2007

Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues. Information

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LANGUAGE: eng

NlmUniqueID: 370623

Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues. Keywords Mesh Terms:

KEYWORDS: Uridine Diphosphate Galactose

MESH TERMS: metabolism

Chemical & Substance for Abstract: Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues. Information

Substance Name: UDP-galactopyranose mutase

Registry Number: EC 5.4.99.9

Grant and Affiliation Information for Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues.

AFFILIATION: Department of Chemistry, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5C9.

Country: United States

AGENCY: United States NIGMS

GRANT: T32 GM008349

ACRONYM: GM

MEDLINETA: Biochemistry

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