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Title: Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
NSFNIH
OSTI Identifier:
1357651
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 55; Journal Issue: 23
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Stull, Frederick W., Bernard, Steffen M., Sapra, Aparna, Smith, Janet L., Zuiderweg, Erik R. P., and Palfey, Bruce A.. Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase. United States: N. p., 2016. Web. doi:10.1021/acs.biochem.6b00510.
Stull, Frederick W., Bernard, Steffen M., Sapra, Aparna, Smith, Janet L., Zuiderweg, Erik R. P., & Palfey, Bruce A.. Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase. United States. doi:10.1021/acs.biochem.6b00510.
Stull, Frederick W., Bernard, Steffen M., Sapra, Aparna, Smith, Janet L., Zuiderweg, Erik R. P., and Palfey, Bruce A.. 2016. "Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase". United States. doi:10.1021/acs.biochem.6b00510.
@article{osti_1357651,
title = {Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase},
author = {Stull, Frederick W. and Bernard, Steffen M. and Sapra, Aparna and Smith, Janet L. and Zuiderweg, Erik R. P. and Palfey, Bruce A.},
abstractNote = {},
doi = {10.1021/acs.biochem.6b00510},
journal = {Biochemistry},
number = 23,
volume = 55,
place = {United States},
year = 2016,
month = 6
}
  • A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M. tuberculosis ThyX (MtbThyX) enzyme complements the E. coli {chi}2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and themore » substrate analog, 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.« less
  • The YES1 proto-oncogene was mapped previously to human chromosome band 18q21.3 by using isotopic in situ hybridization. Using yeast artificial chromosomes (YACs) as probes and fluorescence in situ hybridization, a strong signal was detected in the region corresponding to 18p11.3. Restriction digests confirmed that the YACs contained the YES1 gene and not other cross-hybridizing, protein-tyrosine kinases. In addition, these YACs were found to contain another 18p11.32 gene, thymidylate synthase. These genes were less than 50 kb apart. Collectively, these data suggest that YES1 maps to 18p11.32 rather than to 18p21.3. 18 refs., 3 figs.
  • Activity of the thymidylate synthase cycle was compared in the human ovarian carcinoma cell line A2780 and a subline that is resistant to cisplatin by a factor of 3. Resistant cells exhibited a 3-fold increase in mRNA for both dihydrofolate reductase and thymidylate synthase when compared with the parent line. Resistance to cisplatin also resulted in a 2.5-fold increase in enzyme activity for dihydrofolate reductase and thymidylate synthase; however, this increase did not result from amplification of the genes for these two enzymes. These data suggest that the initial step of cisplatin resistance in A2780 cells is a consequence ofmore » enhanced expression of the thymidylate synthase cycle.« less
  • Allosteric interaction between thymidylate synthase (TS) and the other enzymes of DNA biosynthesis was suggested from the authors observation that inhibitors of ribonucleotide reductase, topoisomerase of DNA polymerase-..cap alpha.. inhibit TS in intact S phase CHEF/18 cells, but not in their soluble extracts. In addition the authors observed that 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), a poison of topoisomerase II, had similar effects on TS activity in mammalian cells. They have examined if the inhibitory effects of these antimetabolites on TS is due to the accumulation of thymidine nucleotide(s) in intact cells, rather than to an allosteric interaction in the replitase complex. A novelmore » method of nucleotide pool analysis revealed that in the presence of these antimetabolites the incorporation of radioactivity from /sup 3/H-deoxyuridine (dUrd) into thymidine nucleotide pools inside the cell did not increase as compared to the control. Furthermore, TS activity as measured in-vitro was not inhibited by supraphysiological concentrations (50..mu..M) of thymidine mono- or tri-phosphates. None of these antimetabolites dramatically influenced the uptake of dUrd and its subsequent phosphorylation to deoxyuridine monophosphate. Therefore, they suggest that the inhibitory effect of these antimetabolites is due to the functional association of their target enzymes with TS.« less