Literature summary for 3.5.4.1 extracted from

Stolworthy, T.S.; Korkegian, A.M.; Willmon, C.L.; Ardiani, A.; Cundiff, J.; Stoddard, B.L.; Black, M.E.
Yeast cytosine deaminase mutants with increased thermostability impart sensitivity to 5-fluorocytosine (2008), J. Mol. Biol., 377, 854-869.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of wild-type and mutant enzymes in Escherichia coli enzyme-deficient strain GIA39(DE3), genetic complementation assays, construction of mammalian expression vectors, overview	Saccharomyces cerevisiae

Crystallization (Commentary)

Crystallization (Comment)	Organism
recombinant A23L/D92E/V108I/I140L mutant bound to the transition state analogue, hanging drop vapour diffusion method, 2 days, crystals are soaked for 20 min in a mother liquor solution containing 2-hydroxypyrimidine concentrated 1.2:1 relative to protein, after soaking, the crystals are immediately transferred briefly to a cryo-buffer containing the 2-hydroxypyrimidine mother liquor plus 25% DMSO, X-ray diffraction structure determination and analysis at 2.3 A resolution, modelling and molecular replacement	Saccharomyces cerevisiae

Crystallization (Comment)

Organism

recombinant A23L/D92E/V108I/I140L mutant bound to the transition state analogue, hanging drop vapour diffusion method, 2 days, crystals are soaked for 20 min in a mother liquor solution containing 2-hydroxypyrimidine concentrated 1.2:1 relative to protein, after soaking, the crystals are immediately transferred briefly to a cryo-buffer containing the 2-hydroxypyrimidine mother liquor plus 25% DMSO, X-ray diffraction structure determination and analysis at 2.3 A resolution, modelling and molecular replacement

Saccharomyces cerevisiae

Protein Variants

Protein Variants	Comment	Organism
A23L/D92E/V108I/I140L	construction of superimposed mutants by combining randomly generated single nucleotide mutants with the triple mutant, the superimposed mutant does not show enhanced activity with 5-fluorouracil and negates the effect introduced by the triple substitutions	Saccharomyces cerevisiae
A23L/I140L	site-directed mutagenesis, the mutant display elevated unfolding temperatures in denaturation experiments and increased half-lives of catalytic activity at elevated temperatures, the mutant cells show an about 30% reduced sensitivity to 5-fluorouracil compared to the wild-type cells	Saccharomyces cerevisiae
A23L/M93L/V108I/I140L	construction of superimposed mutants by combining randomly generated single nucleotide mutants with the triple mutant, the superimposed mutant does not show enhanced activity with 5-fluorouracil and negates the effect introduced by the triple substitutions	Saccharomyces cerevisiae
A23L/V108I/I140L	site-directed mutagenesis, display elevated unfolding temperatures in denaturation experiments and increased half-lives of catalytic activity at elevated temperatures, the mutant cells show an about 50% reduced sensitivity to 5-fluorouracil compared to the wild-type cells	Saccharomyces cerevisiae
A23L/V108I/I140L/I98L	construction of superimposed mutants by combining randomly generated single nucleotide mutants with the triple mutant, the superimposed mutant does not show enhanced activity with 5-fluorouracil and negates the effect introduced by the triple substitutions	Saccharomyces cerevisiae
D92E	random mutagenesis, the mutation is located at the enzyme's dimer interface, the mutant shows increased thermal stability with elevated Tm values and increased activity half-life compared to the wild-type enzyme, the mutant cells show an about 30% reduced sensitivity to 5-fluorouracil compared to the wild-type cells	Saccharomyces cerevisiae
additional information	to create enzyme variants with increased activity to 5-fluorocytosine, 11 codons within the most conserved region of the enzyme, T83, L84, Y85, T86, L88, S89, D92, M93, T95, G96, and I98 are subjected to regiospecific, partially randomizing mutagenesis, Absolutely conserved residues, T87, P90, C91, and C94, within this same region, assumed to be critical for activity, are omitted from randomization, overview	Saccharomyces cerevisiae

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.068	-	5-fluorocytosine	mutant A23L/D92E/V108I/I140L	Saccharomyces cerevisiae
0.083	-	5-fluorocytosine	wild-type enzyme	Saccharomyces cerevisiae
0.14	-	5-fluorocytosine	mutant D92E	Saccharomyces cerevisiae
0.15	-	5-fluorocytosine	mutant A23L/V108I/I140L	Saccharomyces cerevisiae
0.47	-	cytosine	mutant A23L/D92E/V108I/I140L	Saccharomyces cerevisiae
0.52	-	cytosine	mutant A23L/V108I/I140L	Saccharomyces cerevisiae
0.57	-	cytosine	mutant D92E	Saccharomyces cerevisiae
1.17	-	cytosine	wild-type enzyme	Saccharomyces cerevisiae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
5-fluorocytosine + H2O	Saccharomyces cerevisiae	inside a cell, cytosine and 5-fluorocytosine compete for the active site of the enzyme	5-fluorouracil + NH3	-	?
cytosine + H2O	Saccharomyces cerevisiae	the enzyme is a pyrimidine salvage pathway enzyme, inside a cell, cytosine and 5-fluorocytosine compete for the active site of the enzyme	uracil + NH3	-	?

Organism

Organism	UniProt	Comment	Textmining
Saccharomyces cerevisiae	Q12178	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
5-fluorocytosine + H2O	inside a cell, cytosine and 5-fluorocytosine compete for the active site of the enzyme	Saccharomyces cerevisiae	5-fluorouracil + NH3	-	?
5-fluorocytosine + H2O	activation of the prodrug, 5-fluorouracil is an inhibitor of DNA synthesis and RNA function	Saccharomyces cerevisiae	5-fluorouracil + NH3	-	?
cytosine + H2O	-	Saccharomyces cerevisiae	uracil + NH3	-	?
cytosine + H2O	the enzyme is a pyrimidine salvage pathway enzyme, inside a cell, cytosine and 5-fluorocytosine compete for the active site of the enzyme	Saccharomyces cerevisiae	uracil + NH3	-	?

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	in vivo assay at	Saccharomyces cerevisiae

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
9.5	-	cytosine	mutant A23L/D92E/V108I/I140L	Saccharomyces cerevisiae
9.5	-	5-fluorocytosine	mutant A23L/D92E/V108I/I140L	Saccharomyces cerevisiae
18.8	-	5-fluorocytosine	wild-type enzyme	Saccharomyces cerevisiae
29.5	-	cytosine	mutant D92E	Saccharomyces cerevisiae
29.5	-	5-fluorocytosine	mutant D92E	Saccharomyces cerevisiae
31.7	-	cytosine	mutant A23L/V108I/I140L	Saccharomyces cerevisiae
31.7	-	5-fluorocytosine	mutant A23L/V108I/I140L	Saccharomyces cerevisiae
170	-	cytosine	wild-type enzyme	Saccharomyces cerevisiae

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Saccharomyces cerevisiae