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Enzymatic and structural characterization of rTS-gamma provides insights into the function of rTS-beta.

Wichelecki DJ, Froese DS, Kopec J, Muniz JRC, Yue WW, Gerlt JA (2014) Biochemistry 53, 2732-8. PMCID: PMC4010280

In a collaboration with the Structural Genomics Consortium (University of Oxford), the EN Bridging Project characterized human reverse thymidylate synthase (rTS) isoform gamma as a fuconate dehydratase, and determined a catalytic role for a structural component previously proposed to act as a cell localization signal. rTS isoform beta has important implications in a mechanism by which cancer cells exhibit resistance to the chemotherapeutics, 5-fluorouracil and methotrexate. This study exemplifies how the EFI experimental pipeline can be leveraged for functional assignment of human proteins

Abstract

In humans, the gene encoding a reverse thymidylate synthase (rTS) is transcribed in the reverse direction of the gene encoding thymidylate synthase (TS) that is involved in DNA biosynthesis. Three isoforms are found: α, β, and γ, with the transcript of the α-isoform overlapping with that of TS. rTSβ has been of interest since the discovery of its overexpression in methotrexate and 5-fluorouracil resistant cell lines. Despite more than 20 years of study, none of the rTS isoforms have been biochemically or structurally characterized. In this study, we identified rTSγ as an l-fuconate dehydratase and determined its high-resolution crystal structure. Our data provide an explanation for the observed difference in enzymatic activities between rTSβ and rTSγ, enabling more informed proposals for the possible function of rTSβ in chemotherapeutic resistance.

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Abstract Image

Figure 1: Panel A, sequence similarity network for the l-fuconate dehydratase subgroup at an e-value threshold of 10–80 (40% identity). Panel B, network at an e-value threshold of 10–160 (65% identity). Panel C, network at an e-value threshold of 10–180 (70% identity). The nodes for XcFucD (PDB 2HXT) and rTSγ are colored red and blue, respectively.

Figure 2: Structures of the top dehydration screening hits for rTSγ. The first order rate constants for dehydration are shown below the corresponding acid sugar. Carbons with conserved stereochemistry to l-fuconate have their hydroxyl groups highlighted in red.

Figure 3: Panel A, an overlay of the structure of rTSγ (PDB 4A35, tan) and XcFucD bound to the substrate analog L-erythronohydroxamate (PDB 2HXT, blue). The Cα RMSD is 1.03 Å. Panel B shows an overlay of the active site residues of the structures. The Cα RMSD for these metal binding and catalytic residues is 0.31 Å. Residue number is from the structure of rTSγ.

Figure 4: The structure of rTSγ (PDB 4A35) from side and top views. Each structure has the first 27 amino acids, which would be missing in rTSβ, highlighted in red. The metal binding residues are highlighted in green and catalytic residues in cyan.

Reprinted with permission from Biochemistry.
© 2011 American Chemical Society.