Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase Sarathy Karunan Partha 1 †, Ali Sadeghi-Khomami 2,3,4 †, Kathryn Slowski 1 , Toshihisa Kotake 5 , Neil R. Thomas 4 , David L. Jakeman 2,3 and David A. R. Sanders 1 ⁎ 1 Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5C9 2 Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J3 3 College of Pharmacy, Dalhousie University, Burbidge Building, 5968 College Street, Halifax, Nova Scotia, Canada B3H 3J5 4 School of Chemistry, Center for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK 5 Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohubo, Sakura-ku, Saitama 338-8570, Japan Received 25 June 2010; received in revised form 27 August 2010; accepted 30 August 2010 Available online 17 September 2010 Edited by G. Schulz Keywords: UDP-galactopyranose mutase; phosphonate analog; enzyme inhibition and binding UDP (uridine diphosphate) galactopyranose mutase (UGM) is involved in the cell wall biosynthesis of many pathogenic microorganisms. UGM catalyzes the reversible conversion of UDP-α-D-galactopyranose into UDP- α-D-galactofuranose, with the latter being the precursor of galactofuranose (Galf) residues in cell walls. Glycoconjugates of Galf are essential components in the cell wall of various pathogenic bacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. The absence of Galf in humans and its bacterial requirement make UGM a potential target for developing novel antibacterial agents. In this article, we report the synthesis, inhibitory activity, and X-ray crystallographic studies of UDP- phosphono-galactopyranose, a nonhydrolyzable C-glycosidic phosphonate. This is the first report on the synthesis of a phosphonate analog of UDP-α-D- galactopyranose by a chemoenzymatic phosphoryl coupling method. The phosphonate was evaluated against three bacterial UGMs and showed only moderate inhibition. We determined the crystal structure of the phospho- nate analog bound to Deinococcus radiodurans UGM at 2.6 Å resolution. The phosphonate analog is bound in a novel conformation not observed in UGM–substrate complex structures or in other enzyme–sugar nucleotide phosphonate complexes. This complex structure provides a structural basis *Corresponding author. E-mail address: david.sanders@usask.ca. † S.K.P. and A.S.-K. contributed equally to this work. Abbreviations used: UGM, UDP (uridine diphosphate) galactopyranose mutase; Galf, galactofuranose; UDP-Galp, UDP-α-D-galactopyranose; UDP-Galf, UDP-α-D-galactofuranose; FAD, flavin adenine dinucleotide; UDP-CH 2 -Galp, UDP-phosphono-galactopyranose; AtUSP, Arabidopsis thaliana UDP-sugar pyrophosphorylase; ESI, electrospray ionization; MS, mass spectrometry; RT, room temperature; EcUGM, Escherichia coli UGM; UDP-CH 2 -Galf, UDP- phosphono-galactofuranose; DrUGM, Deinococcus radiodurans UGM; KpUGM, Klebsiella pneumoniae UGM; MtUGM, Mycobacterium tuberculosis UGM; GlcNAc, N-acetyl glucosamine; UDP-CH 2 -GlcNAc, UDP-phosphono-GlcNAc; GnT1, N-acetyl glucosaminyl-transferase 1; PDB, Protein Data Bank; OGT, O-linked GlcNAc transferase. doi:10.1016/j.jmb.2010.08.053 J. Mol. Biol. (2010) 403, 578–590 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.