Enzymatic and Structural Characterization of rTSγ Provides Insights into the Function of rTSβ

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.

Novel non-phosphorylative pathway of pentose metabolism from bacteria

“…2b). Three acid-sugar dehydratases that utilize D-arabinonate as the substrate have been identified, but belong to the enolase superfamily; D-arabinonate dehydratase from Archaeon Sulfolobus solfataricus 19 ; L-fuconate dehydratases from Xanthomonas campestris and humans 28,31 . Collectively, these results suggest that the C785_RS13685 protein is a novel D-arabinonate dehydratase enzyme, and that strict substrate specificity may be involved in (unidentified) the physiological metabolic pathway (of D-arabinose, see below).

Figure 2Functional characterization of C785_RS13685 as a D-arabinonate dehydratase.

…”

Proteome-Wide Analysis of Lysine 2-Hydroxyisobutyrylation in the Phytopathogenic Fungus Botrytis cinerea

“…For example, K120 was identified to be 2-hydroxyisobutyrylated in argininosuccinate synthase (Bcass1) ( Supplementary Table 11), and this site is located in the predicted conserved loop of Thr 118 -X-Lys 120 -Gly 121 -Asn 122 -Asp 123 -X-X-Arg 126 -Phe 127 ( Figure 8A) which interacts with the substrates in human and Thermus thermophilus (Goto et al, 2002;Karlberg et al, 2008). Five K hib sites (K157, K173, K177, K194, K262) were identified in B. cinerea L-galactonate dehydratase (Bclgd1), among which Viaud et al, 2003 the K194 is the second K locating in the predicted K × K motif ( Figure 8B) which had been reported to function for base-catalyzed proton abstraction in human (Wichelecki et al, 2014). Interestingly, the lysine 2-hydroxyisobutyrylation was conservative in the conserved loop of Thr-X-Lys-Gly-Asn-Asp-X-X-Arg-Phe in human, rice and Physcomitrella patens (Meng et al, 2017;Yu et al, 2017;Huang et al, 2018; Figure 8C).…”

UniProt: a hub for protein information

“…The ENOSF1 gene coding for this protein was initially thought to code for an antisense RNA to thymidylate synthase, able to down-regulate TYMS activity ( 8 , 9 ). Its function remained largely unknown for many years until recently, with its identification as an L-fuconate dehydratase ( 10 ). L-fuconate dehydratase is involved in catabolism of L-fucose, a sugar that is part of the carbohydrates that are attached to cellular glycoproteins, and catalyzes the dehydration of L-fuconate to 2-keto-3-deoxy-L-fuconate.…”

“…A lot of sequence annotation information is derived from 3D structures, leveraging information from the Protein Data Bank (PDB) in combination with information extracted from the papers. In the case of ENOSF1 (UniProt Q7L5Y1), a high-resolution crystal structure in complex with magnesium cofactor is available, allowing the annotation of cofactor binding sites on the sequence ( 10 ). A crystal structure in complex with magnesium and substrate is available for X. campestris fuconate dehydratase (UniProt Q8P3K2), allowing the reliable propagation of the substrate binding sites to the human entry, thanks to the high sequence conservation of around 50% between the two enzymes (Figure 3 ).…”