Purification and characterization of RihC, a xanthosine-inosine-uridine-adenosine-preferring hydrolase from Salmonella enterica serovar Typhimurium
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Purification and characterization of RihC, a xanthosine-inosine-uridine-adenosine-preferring hydrolase from Salmonella enterica serovar Typhimurium. / Hansen, Michael Riis; Dandanell, Gert.
I: Biochimica et Biophysica Acta - General Subjects, Bind 1723, Nr. 1-3, 2005, s. 55-62.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Purification and characterization of RihC, a xanthosine-inosine-uridine-adenosine-preferring hydrolase from Salmonella enterica serovar Typhimurium
AU - Hansen, Michael Riis
AU - Dandanell, Gert
N1 - Keywords: Nucleoside hydrolase; Xanthosine metabolism; Purine salvage pathway; pH dependence; Salmonella typhimurium
PY - 2005
Y1 - 2005
N2 - Salmonella enterica serovar Typhimurium normally salvage nucleobases and nucleosides by the action of nucleoside phosphorylases and phosphoribosyltransferases. In contrast to Escherichia coli, which catabolizes xanthosine by xanthosine phosphorylase (xapA), Salmonella cannot grow on xanthosine as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 kDa that can hydrolyze xanthosine, inosine, adenosine and uridine with similar catalytic efficiency (kcat/Km=1 to 4×104 M-1s-1). Cytidine and guanosine is hydrolyzed with approximately 10-fold lower efficiency (kcat/Km=0.7 to 1.2×103 M-1s-1) while RihC is unable to hydrolyze the deoxyribonucleosides thymidine and deoxyinosine. The Km for all nucleosides except adenosine is in the mM range. The pH optimum is different for inosine and xanthosine and the hydrolytic capacity (kcat/Km) is 5-fold higher for xanthosine than for inosine at pH 6.0 while they are similar at pH 7.2, indicating that RihC most likely prefers the neutral form of xanthosine.
AB - Salmonella enterica serovar Typhimurium normally salvage nucleobases and nucleosides by the action of nucleoside phosphorylases and phosphoribosyltransferases. In contrast to Escherichia coli, which catabolizes xanthosine by xanthosine phosphorylase (xapA), Salmonella cannot grow on xanthosine as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 kDa that can hydrolyze xanthosine, inosine, adenosine and uridine with similar catalytic efficiency (kcat/Km=1 to 4×104 M-1s-1). Cytidine and guanosine is hydrolyzed with approximately 10-fold lower efficiency (kcat/Km=0.7 to 1.2×103 M-1s-1) while RihC is unable to hydrolyze the deoxyribonucleosides thymidine and deoxyinosine. The Km for all nucleosides except adenosine is in the mM range. The pH optimum is different for inosine and xanthosine and the hydrolytic capacity (kcat/Km) is 5-fold higher for xanthosine than for inosine at pH 6.0 while they are similar at pH 7.2, indicating that RihC most likely prefers the neutral form of xanthosine.
U2 - 10.1016/j.bbagen.2005.01.012
DO - 10.1016/j.bbagen.2005.01.012
M3 - Journal article
VL - 1723
SP - 55
EP - 62
JO - B B A - General Subjects
JF - B B A - General Subjects
SN - 0304-4165
IS - 1-3
ER -
ID: 94777