Inhibition of Escherichia coli CTP Synthetase by NADH and Other Nicotinamides and Their Mutual Interactions with CTP and GTP

Chris Habrian, Adithi Chandrasekhara, Bita Shahrvini, Brian Hua, Jason Lee, Roger Jesinghaus, Rachael Barry, Zemer Gitai, Justin Kollman, Enoch P. Baldwin

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

CTP synthetases catalyze the last step of pyrimidine biosynthesis and provide the sole de novo source of cytosine-containing nucleotides. As a central regulatory hub, they are regulated by ribonucleotide and enzyme concentration through ATP and UTP substrate availability, CTP product inhibition, GTP allosteric modification, and quaternary structural changes including the formation of CTP-inhibited linear polymers (filaments). Here, we demonstrate that nicotinamide redox cofactors are moderate inhibitors of Escherichia coli CTP synthetase (EcCTPS). NADH and NADPH are the most potent, and the primary inhibitory determinant is the reduced nicotinamide ring. Although nicotinamide inhibition is noncompetitive with substrates, it apparently enhances CTP product feedback inhibition and GTP allosteric regulation. Further, CTP and GTP also enhance each other's effects, consistent with the idea that NADH, CTP, and GTP interact with a common intermediate enzyme state. A filament-blocking mutation that reduces CTP inhibitory effects also reduced inhibition by GTP but not NADH. Protein-concentration effects on GTP inhibition suggest that, like CTP, GTP preferentially binds to the filament. All three compounds display nearly linear dose-dependent inhibition, indicating a complex pattern of cooperative interactions between binding sites. The apparent synergy between inhibitors, in consideration with physiological nucleotide concentrations, points to metabolically relevant inhibition by nicotinamides, and implicates cellular redox state as a regulator of pyrimidine biosynthesis.

Original languageEnglish (US)
Pages (from-to)5554-5565
Number of pages12
JournalBiochemistry
Volume55
Issue number39
DOIs
StatePublished - Oct 4 2016

All Science Journal Classification (ASJC) codes

  • Biochemistry

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    Habrian, C., Chandrasekhara, A., Shahrvini, B., Hua, B., Lee, J., Jesinghaus, R., Barry, R., Gitai, Z., Kollman, J., & Baldwin, E. P. (2016). Inhibition of Escherichia coli CTP Synthetase by NADH and Other Nicotinamides and Their Mutual Interactions with CTP and GTP. Biochemistry, 55(39), 5554-5565. https://doi.org/10.1021/acs.biochem.6b00383