Characterization of a novel resistance-related deoxycytidine deaminase from Brassica oleracea var. capitata.
Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2'-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2'-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine β-d-arabinofuranoside than in deaminating 2'-deoxycytinde to 2'-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 °C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 μM and 1.475 mM for its natural substrate 2'-deoxycytidine and 63 μM and 2.072 mM for cytosine β-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties.
Department of Life Science and the Institute of Biotechnology, National Dong Hwa University , Hualien 97401, Taiwan, Republic of China.
Marthandam Asokan
MA
Shibu
Hsueh-Hui
HH
Yang
Chaur-Tsuen
CT
Lo
Hong-Shin
HS
Lin
Shu-Ying
SY
Liu
Kou-Cheng
KC
Peng
D001937:Brassica / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003562:Cytidine / Q000378:metabolism; D003564:Cytidine Deaminase; D003596:Cytosine / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009700:Nucleoside Deaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014529:Uridine / Q000378:metabolism
D010940:Plant Proteins; D003562:Cytidine; D003596:Cytosine; D009700:Nucleoside Deaminases; D003564:Cytidine Deaminase; C011530:deoxycytidine deaminase; D014529:Uridine