Hydrolysis of the 5'-p-nitrophenyl ester of TMP by oligoribonucleases (ORN) from Escherichia coli, Mycobacterium smegmatis, and human.
Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3'-5' exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E. coli, but not in higher organisms, and close homologues are present in other genomes from the beta and gamma subdivisions of the Protobacteriaceae, including many pathogenic species. We report here the expression in E. coli of orn and homologues from Mycobacterium smegmatis and human, and large-scale purification of the three enzymes. All three were found to promote the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of Michaelis-Menten parameters (k(cat)=100-650 min(-1), K(M)=0.4-2.0 mM, at pH 8.00 and 25 degrees C, with 1 mM Mn(2+)). Hydrolysis by pNP-TMP by all three enzymes depended on a divalent metal ion, with Mn(2+) being preferred over Mg(2+) as cofactor, and was inhibited by Ni(2+). The concentration dependency of Mn(2+) was examined, giving K(Mn) values of 0.2-0.6 mM. The availability of large amounts of the purified enzymes and a simple spectrophotometric assay for ORN activity should facilitate large-scale screening for new inhibitors of bacterial oligoribonucleases.
Young Park
Ah
A
Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
Elvin
Christopher M
CM
Hamdan
Samir M
SM
Wood
Robert J
RJ
Liyou
Nancy E
NE
Hamwood
Tamarind E
TE
Jennings
Phil A
PA
Dixon
Nicholas E
NE
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D005095:Exoribonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D009596:Nitrophenols / Q000378:metabolism*; D009842:Oligopeptides; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D012995:Solubility; D013938:Thymidine Monophosphate / Q000378:metabolism*
C471213:His-His-His-His-His-His; D009596:Nitrophenols; D009842:Oligopeptides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D013938:Thymidine Monophosphate; D006639:Histidine; D005095:Exoribonucleases; C094587:oligoribonuclease