Arabidopsis thaliana Cytidine Deaminase 1 Shows More Similarity to Prokaryotic Enzymes Than to Eukaryotic Enzymes

Kafer, Chris,Thornburg, Robert W. 한국식물학회 2000 Journal of Plant Biology Vol.43 No.3

Two Expressed Sequence Tagged (EST) clones were identified from the Arabidopsis 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 acid 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 27% 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 His_6 tag. Following purification on a Ni-NTA-Agarose column, the protein was analyzed for its kinetic properties. The enzyme utilizes both cytidine (K_m= 226μM) and 2'-deoxycytidine (K_m=49μM) as substrates. The enzyme was unable to deaminate cytosine, CMP or dCMP.

Molecular Cloning of Bacillus stearothermophilus cdd Gene Encoding Thermostable Cytidine/Deoxycytidine Deaminase

Soo, Chang-Jong,Song, Bang-Ho,Kim, Jong-Guk,Hong, Soon-Duck 한국미생물 · 생명공학회 1989 한국미생물·생명공학회지 Vol.17 No.4

Bacillus stearothermophilus의 cytidine deaminase (cytidine/2'-deoxycytidine aminohydrolase:EC 3.5.4.5)를 코딩하는 cdd 유전자를 E. coli cdd$^-$ 결손변이주를 cloning host로 하여 3-10Kbp의 B. stearothermophilus DNA 단편으로부터 shot gun 법으로 클로닝하였다. 고 복제수 플라스미드 pBR322 의 PstI 부위에 3.0Kb의 B. stearothermophilus DNA 단편을 함유한 pJSC101이 cdd$^+$와 tetracy-line 내성으로서 cloning되었으며, 이어서, 결실 및 subcloning을 연속 수행한 결과 약 1.35kbp의 Eco RI$_1$/PstI$_2$단편이 동일 부위의 pBR322에 삽입된 cdd 양성의 pJSC201을 얻었다. Mini 세포 실험결과, 이 단편에서 합성되는 polypeptide는 약 33 KDa이었기에 이 polypeptide가 cytidine deaminase 로 추정되었다. 또한 이 단편에 함유한 550bp의 EcoRI/AvaI 부분을 lacZ 프로모터 영역에 삽입한 경우 프로모터 활성을 나타내었기에 이 단편의 Eco RI 부위에서 PstI부위로 cdd 유전자가 전사됨을 알 수 있었다. B. subilis와 E. coli에서 발현이 가능한 shuttle vector에 cdd가 함유된 단편을 삽입한 후 이를 양세포에서 동시 발현시켰을 때 B. subtilis에서 발현시킨 경우가 E. coli에서 보다높은 cytidine deaminase 활성을 나타내었으며 이 유전자는 B. subtilis 에서도 E. coli에서와 같이 안정하게 유지됨을 알 수 있었다. The Bacillus stearothermophilus cdd gene encoding cytidine deaminase (cytidine/2'-deoxycytidine aminohydrolase; EC 3.5.4.5) was isolated through shot gun cloning by oomplementation of an E. coli cdd mutation. Primarily 3.0 kbp of the exogenote was cloned into the Pstl site of pBR322 (pJSC101). By subsequent deletion and subcloning from the insert of pJSC101 with cdd$^+$ and tetracycline resistancy, about 1.35 kbp of the EcoRI$_1$/PstI$_2$ fragment containing the cdd gene was isolated as pJSC201. The minicell experiment revealed a molecular mass of 33,000 dalton for polypeptide from the cloned DNA fragment complementing the cdd gene. From the lacZ fusion of 550 bp fragment of the EcoRI$_1$/AuaI as a putative promoter region, the transcription direction of the cdd gene on pJSC201 is from EcoRI towards the PstI sites, When the cdd gene was expressed in B. subtilis ED4O (cdd$^-$, pyr$^-$) by transformation with the E. coli-B. subtilis shuttle vector, the gene expression occured more efficiently than in E. coli and the gene appears to be stably maintained in B. subtitis as well as in E. coli.

화학적수식에 의한 Bacillus subtilis ED 213 Cytidine Deaminase의 활성부위에 관한 연구

박정문,박상원,서태수,김정,유대식,Park, Jung-Moon,Park, Sang-Won,Suh, Tae-Soo,Kim, Jung,Yu, Tae-Shick 한국미생물학회 1999 미생물학회지 Vol.35 No.2

Essential amino acids involving in the active site ofthe cytidn~e deruninase from Bncillus subtilis ED 213 were determined by chemical modification studies. Tllc purified cytidine deruninase tiom Booillus subtilis ED 213 required the reduced form of Fe(lI)ion. since the enzyme was inhibited 43% by 1 mnM o-phenanthroline. Whereas the enzyme activity was activated up to 28% by 1 1 ethylenediaminetetraacetic acid. The cytidine deaninase activily was completely inhibited by 1 mM N-bromosuccinimide, chloramine-T, and p-chloromercuribenzoic acid (p-CMB), respectively. The enzyme activity was inhibited 36% by 1 mM pyridoxal-S-phosphale, and 31% by 1 mM l-ethy~-3-(3-dirneIhj~laminoprop}~~)c~bodiiamide and glycine inethyl ester. The enzyme activity was strongly inhibited 68% by 1 \mu$M \rho$-CMB and this inhibition of the enzyme activity with 1 \mu$M \rho$-CMB was completely reactivated by 5 mM cysleine as a reducing agent. We speculaled that tyrosine, methionine, cysteuie and/or serine residues are located ui or near ihe active site of the cytidine deruniuase from Bncilus subrilis ED 213 and indirectly related to lysine and/or glycine. Bacillus subtilis ED 213의 cytidine deaminase 의 활성부위에 존재하는 필수 아미노산잔기를 화학수식 방법으로 측정하였다. 본 효소는 1mM o-phenanthroline 에 의하여 효소활성이 43% 저해되어 효소활성 발현에 Fe\sup 2+\가 요구된다고 추정되며, 1mM ethylenediaminetetraacetic acid 에 의해서는 효소활성이 오히려 28% 정도 촉진되었다. 본 효소는 1mM N-bromosuccinimide, 1mM chloramine-T 와 1mM $\rho$-chloromercuribenzoic acid에 의하여 100% 저해되었으며, 그의 저해 양상은 경쟁적 저해 양상을 나타내었다. 본 효소의 효소활성은 1mM pyridoxal-5-phosphate 에 의항 36% 저해되었으며, 1mM 1ethyl-3-carbodiamide 와 1mM glycine methylester에 의해 저해된 효소활성이 5mM cysteine에 의해 완전히 회복되었다. 이상의 결과로부터 Bacillus subtilis ED 213 cytidine deaminase의 활성부위에는 tyrosine, methionine, cysteine 과 serine 잔기가 관여할 뿐만 아니라 lysine 과 glycine 도 효소활성에 관여하는 것으로 추정된다.

Secretion of Bacillus subtilis Cytidine Deaminase by the Aid of Signal Sequences in Escherichia coli

Yoon, Soo Ryun,Kim, Sung Il,Lee, Se Young,Song, Bang Ho 한국미생물 · 생명공학회 1991 Journal of microbiology and biotechnology Vol.1 No.1

In order to secrete the Bacillus subtilis cytidine deaminase (CDase, cytidine/2'-deoxycytidine deaminase) encoded by the B. subtills cdd gene in E, coli by the aid of signal sequences, the cdd gene was fused in-frame to either amyE or penP signal sequences and the gene expression and CDase localization were examined. For the penP signal sequence::cdd fusion, the cdd gene with 9 amino acids truncated from the 5'-terminus was fused in-frame to the signal sequence, then the cdd^+ colonies were not occurred from the minimal plate by cdd complementation. The result suggests that 9 amino acids on the NH_2-terminal of CDase have an essential function in the enzyme activity. The hybrid protein obtained by fused gene amyE signal sequence::cdd structural gene gave cdd^+ phenotype and about half of the total CDase activity was found to be secreted in the periplasm of E. coli transformant JF611/pSO202. The periplasmic CDase activity of JF611 harboring pSO52 containing the intact cdd gene was considerablely lower than that of the cells harboring pSO202 carrying the hybrid cdd gene. This suggests that the CDase was secreted to the periplasm through the cytoplasmic membrane by the aid of the amyE signal sequence in the E. coli transformant.

Escherichia coli의 시티딘/디옥시시티딘 디아미나제를 코드하는 cdd 유전자의 클로닝

권택규,김태호,황선갑,김종국,송방호,홍순덕 한국산업미생물학회 1990 한국미생물·생명공학회지 Vol.18 No.6

E. coli의 cytidine deaminase(cytidine/2'-deoxycytidine aminohydrolase; EC 3.5,4.5)를 코딩하는 cdd 유전자를 E. coli cdd^-pyr^- 결손 변이주를 cloning host로 하여 southern blotting과 colony hybridization을 통하여 클로닝하였다. cdd 유전자가 단편은, cdd 유전자의 transcription initiation 부위의 23개 nucleotide를 합성한 후 probe로 사용하여 Southern hybridization에 의해 회수된 cdd 유전자를 함유한 단편을 얻었으며, 이를 pBR322에 삽입한 후 형질전환하여 colony hybridization한 결과 cdd^+ cell을 얻었다. 삽입된 DNA 단편의 size는 27kb이었으며 이를 결실 및 subcloning을 연속 수행한 결과 2.1kb의 SalI/DraI tragment(pTK605)에 cdd 유전자가 location되어 있음을 알게 되었다. Mini cell 실험결과 합성된 polypeptide는 약 33kDa이었으며, wild type의 cytidine deaminase의 활성이 pBR322에서 증폭시킴으로서 37배 정도 배가되었으며, pBR322에 비해 pUC vector계에서 다시 활성이 7배 정도 증가됨을 알 수 있었다. We have cloned the cdd gene from E. coli C600 using (cdd^-) as a host. From the sequenced promoter region of E. coli cdd gene which has been determined by Valentin-Hansen P. (1985), we synthesized the 23 mer oligonucleotides corresponding to the transcription initiation region and used as a probe for cloning of the cdd gene by Southern blotting. The isolated fragments in the blotting were introduced to the colony hybridization after transforming it into the E. coli JF611 (cdd^-, pyr^- double mutant), and we identified the hybridized band at 27 kb long. From the original insert of 27 kb fragment in the BamHI site of pBR322, the 5.3 kb fragment containing the cdd gene was isolated by subsequent deletion and subcloning. From the derived plasmid pTK509, further deletion and subcloning were performed and clarified that the cdd gene was located in the 2.1 kb of SalI/DraI segment in the insert of pTK605. The polypeptide encoded by the cloned DNA was appeared to be a molecular mass of 33,000.

Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST

Tong, Yaojun,Whitford, Christopher M.,Robertsen, Helene L.,Blin, Kai,Jørgensen, Tue S.,Klitgaard, Andreas K.,Gren, Tetiana,Jiang, Xinglin,Weber, Tilmann,Lee, Sang Yup National Academy of Sciences 2019 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.116 No.41

<P><B>Significance</B></P><P>Although CRISPR-Cas9 tools dramatically simplified the genetic manipulation of actinomycetes, significant concerns of genome instability caused by the DNA double-strand breaks (DSBs) and common off-target effects remain. To address these concerns, we developed CRISPR-BEST, a DSB-free and high-fidelity single-nucleotide–resolution base editing system for streptomycetes and validated its use by determining editing properties and genome-wide off-target effects. Furthermore, our CRISPR-BEST toolkit supports Csy4-based multiplexing to target multiple genes of interest in parallel. We believe that our CRISPR-BEST approach is a significant improvement over existing genetic manipulation methods to engineer streptomycetes, especially for those strains that cannot be genome-edited using normal DSB-based genome editing systems, such as CRISPR-Cas9.</P><P>Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide–resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different <I>Streptomyces</I> species. Particularly in nonmodel actinomycete <I>Streptomyces collinus</I> Tu¨365, CRISPR-cBEST efficiently inactivated the 2 copies of <I>kirN</I> gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.</P>

The Enzymatic Characteristics of the Cytidine Deaminase in Salmonella typhimurium

( Sang Mahn Lee ) 한국환경생물학회 2003 환경생물 : 환경생물학회지 Vol.21 No.1

N/A The cytidine deaminase waa partialy purified with sephadex 6-200 and the characteristics of the enzyme were clarified. The moleeular mass of the plasmid-enco-ded protein was identified as about 3OkDa in a minicell system. The native enzyme was estimated to have the molecular mass of 6OkDa by gel filtration. This indicates that the native enzyme may exist as aher composed of two identical subunits. The enzyme was reasonably stable in the pH range of 6 to 9, and wa8 labile under high temperature above 50℃. Mercaptoetbanol, pCMB, mercury and copper were found to inhibit the enzyme activity. The cytidine analogues of bmmo- and iodo-(deoxy)-ytidine were also found to inhibit the activity, while fluomdeoxycytidine and azacytidine were found to activate it. Deoxycytidine, cytidine, ara-C and Methyldeoxycytidine have excellent substrates for the enzyme.

Optimization of Culture Conditions and Analysis fo Plasmid Stability of a Transformant Bacillus subtilis for Cytidine Deaminase Production

Kim, Soo Hyun,Song, Bang ho,Lee, Yong Hyun 한국미생물 · 생명공학회 1991 Journal of microbiology and biotechnology Vol.1 No.2

The transformant Bacillus subtilis ED213 carrying the pSO100 which cloned the cdd gene encoding cytidine deaminase (cytidine /2'-deoxycytidine aminohydrolase, EC 3.5.4.5, CDase) originated from wild type B. subtilis was cultivated in Spizizen minimal medium (SMM). To overcome poor expression of the cdd gene in SMM medium, the medium compositions and growth conditions were optimized. The optimized medium compositions and growth conditions were cytidine concentration of 80㎎/ℓ, glycerol of 25g/ℓ, and (NH_4)_2SO_4 of 10g/ℓ, along with 37℃ and pH 7.0. The intracellular CDase production was increased 3 times from 1,000 unit/㎖ to 3,200 unit/㎖, and extracellular CDase also increased from nearly undetectable amounts to 1,500 unit/㎖. The cytidine concentration was signified as the most critical compositional factor for overproduction of CDase by increasing the cell density mainly in culture broth. The plasmids were more stable in cells that were grown in original SMM medium with stability of 90% compared to those grown in optimized SMM medium with stability of 80% after 48 hours cultivation. The most active amplification of plasmid was occurred in the logarithmic phase, which showed a value around four times higher than the initial copy number. In the exponential phase, the CDase production was closely related to the plasmid copy number along with the cell density. However, it was not accorded with cell density at the stationary phase.

thermostability of Chimeric Cytidine Deaminase Variants Produced by DNA Shuffling

Yu Mi Park,Quyet Tien Phi,Bang Ho Song,Sa Youl Ghim 한국미생물 · 생명공학회 2009 Journal of microbiology and biotechnology Vol.19 No.12

Activation-induced Cytidine Deaminase in B Cell Immunity and Cancers

Park, Seok-Rae The Korean Association of Immunobiologists 2012 Immune Network Vol.12 No.6

Activation-induced cytidine deaminase (AID) is an enzyme that is predominantly expressed in germinal center B cells and plays a pivotal role in immunoglobulin class switch recombination and somatic hypermutation for antibody (Ab) maturation. These two genetic processes endow Abs with protective functions against a multitude of antigens (pathogens) during humoral immune responses. In B cells, AID expression is regulated at the level of either transcriptional activation on AID gene loci or post-transcriptional suppression of AID mRNA. Furthermore, AID stabilization and targeting are determined by post-translational modifications and interactions with other cellular/nuclear factors. On the other hand, aberrant expression of AID causes B cell leukemias and lymphomas, including Burkitt's lymphoma caused by c-myc/IgH translocation. AID is also ectopically expressed in T cells and non-immune cells, and triggers point mutations in relevant DNA loci, resulting in tumorigenesis. Here, I review the recent literatures on the function of AID, regulation of AID expression, stability and targeting in B cells, and AID-related tumor formation.