Nowadays, for identification of the function and structure prediction of theprotein being product of new gene, comparison of sequence homology by computational analysis between this new and already known protein seems to be useful tool. These works are mainly based on the conservation of structure concerning the divergent sequence and on the insight of the similar catalytic activity over various substrates. Based on the analysis of conservation pattern in the structure and similarity in the catalytic activity of the enzymes, we have proposed a cyclic amidohydrolase family, including D-hydantoinase, dihydropyrimidinase, dihydro-orotase, allantoinase. We found a number of highly conserved regions and amino acid residues. It is suggested that in archaea, there were existences of thermostable counterparts to known classes enzymes previously characterized from mesophilic sources, which may be concerned to same crucial enzyme reaction in vivo. There was some trial to detect an additional member of amidohydrolase family by computational analysis. Therefore, we attempt to find the new enzyme belonging to this family from the partial available genome databases of Pyrococcus furiosus by using of the homology epitope deduced from amidohydrolase family. Based on these four regions, we probed the whole genome sequence of P.furiosus and led to isolation of 1.5kb ORF fragment encoding putative enzyme, PNT. The ORF encoding putative enzyme was obtained from genomic DNA of P.furiosus, and this 1.5kb fragment was cloned into the plasmid vector pTrc99A, pMAL via EcoRI, PstI restriction sites. and expressed in E Coli. The expressed protein was isolated and its enzymatic activity was tested. Based on the similarities in the conserved region between putative enzyme and member of the amidohydrolase family, we suggested an evolutionary relationship and functional identification
몇 년 전 bacterial genome sequence가 완전히 밝혀진 이후 현재까지 50개 이상의 genome progect들이 진행되고 있고, 가까운 미래에 이루어 질 것을 기대하고 있다. 이런 완전한 genome sequence의 information을 쉽고 빠르게 얻을 수 있는 database가 잘 구축되어 있다. Genome sequence에 대한 정보를 쉽게 얻을 수 있게 된 후 sequence의 comparison을 통해 interesting gene에 대해 기능적으로 관련이 있는 genes을 찾는 것이 가능해졌다. 하지만 이런 sequence에만 기초해서 putactive protein을 찾고 그 특성을 규명하는 데는 많은 제약이 따르고 실제 sequence homology가 낮을 경우 목적 gene을 찾는 것은 그리 쉬운 일이 아니었다. Structure에서의 conservation pattern과 catalytic activity의 유사성에 기인해서 cyclic amidohydrolase family가 제안되었다. 기능적으로 관련이 있는 D-hydantoinase, dihydropyrimidinase, dihydroorotase, allantoinase는 cyclic amide bond를 hydrolysis시키고 sequence 상에서도 유사성이 발견되고 있다. Cyclic amidohydrolase들의 amino acids을 alignment함으로써 highly conserved region과 conserved metal binding residues를 발견할 수가 있었다. Highly conserved region을 가지고 amidohydrolase family의 새로운 member를 찾기 위해 hyperthermophilc archaea인 Pyrococcus furiosus의 genome sequence를 찾게 되었다. Conserved region과 유사성이 보이는 1.5kb의 ORF fragment를 찾아서 expression vector에 cloning시켰다. 그 후 Expression host인 E. coli에서 발현시켜 정제한 후 enzymatic activity를 확인하였다. Putative protein과 amidohydrolase family 사이의 conserved region에서의 유사성에 기인해서 evolutinal relationship과 functional prediction을 제안했다.
