|
|
|
한국식물학회 / v.43, no.3, 2000년, pp.162-170 
|
( Arabidopsis thaliana Cytidine Deaminase 1 Shows More Similarity to Prokaryotic Enzymes Than to Eukaryotic Enzymes ) |
| ;; ;;
|
|
|
 |
|
| |
| 초 록 |
|
|
| Two Expressed Sequence Tagged (EST) clones were identified from the Avabidopsis database as encoding putative cytidine deaminases. Sequence analysis determined that the two clones overlapped and encoded a single cDNA. This cytidine deaminase corresponds to the Arabidopsis thaliana gene, cda1. The deduced amino arid sequence was more closely related to prokaryotic cytidine deaminases than to eukaryotic enzymes. The cDNA shares 44% amino acid identity with the Escherichia coli cytidine deaminase but only 26 and 2Pt identity with human and yeast enzymes. A unique zinc-binding domain of the E. coli enzyme forms the active site. A similar putative zinc-binding domain was identified in the Arabidopsis enzyme based upon primary sequence similarities. These similarities permitted us to model the active site of the Arabidopsis enzyme upon that of the E. coli enzyme. In this model, the active site zinc is coordinated by His$^{73}$ , Cys$^{103}$ , Cys$^{107}$ , and an active site hydroxyl. Additional residues that participate in catalysis, Asn$^{64}$ , Glu$^{66}$ , Ala$^{78}$ , Glu$^{79}$ , and Pro$^{102}$ , are conserved between the Arabidopsis and E. coli enzymes suggesting that the Arabidopsis enzyme has a catalytic mechanism similar to the E. coli enzyme. The two overlapping ESTs were used to prepare a single, full-length clone corresponding to the A. thaliana cda1 cDNA. This cDNA was subcloned into pProExHtb and expressed as a fusion protein with an N-terminal His6 tag. Following purification on a Ni-NTA-Agarose column, the protein was analyzed for its kinetic properties. The enzyme utilizes both cytidine (K$_{m}$ = 226 $mu$M) and 2'-deoxycyti-dine (K$_{m}$ = 49 $mu$M) as substrates. The enzyme was unable to deaminate cytosine, CMP or dCMP.CMP. |
| |
| 키워드 |
| cytidine deaminase; |
| |
|
|
|
|
Journal of Plant Biology / v.43, no.3, 2000년, pp.162-170
한국식물학회
ISSN : 1226-9239
UCI : G100:I100-KOI(KISTI1.1003/JNL.JAKO200011921106671)
언어 : 영어 |
|
| 논문 제공 : KISTI 한국과학기술정보연구원 |
|
|
|
|
|
|
