Mutational analysis of conserved glycine residues 142, 143 and 146 reveals Gly142 is critical for tetramerization of CTP synthase from Escherichia coli

Abstract

CTPS (cytidine 5'-triphosphate synthase) catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source. Binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers. In the present study, site-directed mutagenesis was used to replace the fully conserved glycine residues 142 and 143 within the UTP-binding site and 146 within the CTP-binding site of Escherchia coli CTPS. CD spectral analyses of wild-type CTPS and the glycine mutants showed a slight reduction of approximately 15% in alpha-helical content for G142A and G143A relative to G146A and wild-type CTPS, suggesting some local alterations in structure. Relative to wild-type CTPS, the values of k(cat)/K(m) for ammonia-dependent and glutamine-dependent CTP formation catalysed by G143A were reduced 22- and 16-fold respectively, whereas the corresponding values for G146A were reduced only 1.4- and 1.8-fold respectively. The glutaminase activity (k(cat)) of G146A was similar to that exhibited by the wild-type enzyme, whereas that of G143A was reduced 7.5-fold. G146A exhibited substrate inhibition at high concentrations of ammonia and a partial uncoupling of glutamine hydrolysis from CTP production. Although the apparent affinity (1/[S](0.5)) of G143A and G146A for UTP was reduced approximately 4-fold, G146A exhibited increased co-operativity with respect to UTP. Thus mutations in the CTP-binding site can affect UTP-dependent activity. Surprisingly, G142A was inactive with both ammonia and glutamine as substrates. Gel-filtration HPLC experiments revealed that both G143A and G146A were able to form active tetramers in the presence of ATP and UTP; however, nucleotide-dependent tetramerization of G142A was significantly impaired. Our observations highlight the sensitivity of the structure of CTPS to mutations in the UTP- and CTP-binding sites, with Gly(142) being critical for nucleotide-dependent oligomerization of CTPS to active tetramers. This 'structural sensitivity' may limit the number and/or types of mutations that could be selected for during the development of resistance to cytotoxic pyrimidine nucleotide analogues.

Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation*

“…Activity of both hCTPS1 and hCTPS2 was regulated by oligomerization as seen with other CTPS enzymes (8,16,25,38). Tetramerization can be induced by ATP and/or UTP, and at high enzyme concentrations equilibrium often exists between monomers, dimers, and tetramers (29).…”

“…Similar to hCTPS, ATP or UTP alone can induce the formation of tetramers in E. coli (25). However, in contrast to hCTPS, ATP and UTP synergistically stimulate tetramer formation in E. coli (16). Interestingly, the pattern of elution of hCTPS1 appears to be more complex than simple monomers, dimers, and tetramers.…”

“…Although elegant analysis of the enzymatic properties of the E. coli and yeast enzymes has been performed (4,8,12,15,16,25,28), the individual human isoforms have not been examined in detail. The objective of this study was to explore the regulation of hCTPS1 and hCTPS2 enzyme activity by substrates, modulators, and phosphorylation as well as to determine the requirement for ATP and/or UTP for enzyme tetramerization.…”

Common regulatory control of CTP synthase enzyme activity and filament formation

“…, 2004). In the Escherichia coli enzyme, a mutation in the binding site of the substrate UTP, which lies in the amidoligase domain near the tetramer interface, has been shown to severely compromise UTP binding, tetramer formation and catalytic activity (Figure 2B; Lunn et al. , 2008).…”

Molecular characterization of a novel trehalose-6-phosphate hydrolase, TreA, from Bacillus licheniformis

“…Protein oligomerization was reported as a concentration-dependent event and increased logarithmically with increasing concentration [58]. Therefore, many proteins were demonstrated to have an interconvertible mixture of monomers and oligomers [59][60][61]. Although Flavobacterium sp., T. vulgaris, and P. furiosus enzymes have been described as dimers in the past, it is still not clear whether the family 13 enzymes form active monomers or dimers, or what is the physiological role of a possible oligomerization state.…”