Sphingomonas chungbukensis DJ77 균주에서Phosphomannomutase를 암호화하는 pmmC 유전자의 클로닝과 발현
Expression and Cloning of the pmmC Gene Encoding Phosphomannomutase in Sphingomonas chungbukensisDJ77.
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초록
Phosphomannomutase는 진핵 생물과 원핵 생물에 있어서중요한 효소로, α-D-mannose 6-phosphate를 α-D-mannose1-phosphate로 전환시켜 GDP-mannose를 생산한다. 이 기질은여러 대사 경로에서 중요하게 작용하는 mannosyl기를 제공하도록 돕는다. 본 논문에서는 Sphingomonas chungbukensisDJ77에서 phosphomannomutase를 암호화하는 유전자를 유전체 library로부터 동정하고 이를 pmmC로 명명하였으며,이를 발현 vector에 클로닝하고 염기서열을 분석하였다. 유전자 pmmC는 ATG를 개시 코돈으로 사용하고, TAG를 종결 코돈으로 사용하는 750 bp의 open reading frame임을 확인하였고, 이 ORF의 5 bp 앞쪽으로 리보좀 결합 부위가 존재한다. 이 ORF로부터 유추되는 아미노산은 249개이며, 단백질 분자량은 약 27.4 kDa이다. 이 유전자를 구성하는 아미노산 서열은 NCBI의 conserved domain search를 통한분석으로 eukaryotic phosphomannomutase와 약 86.9% 유사성이 있음을 나타냈고, 기질에 대한 활성을 측정한 결과pmmC 유전자가 암호화하는 단백질이 phosphomannomutase임을 확인할 수 있었다.
Abstract
Phosphomannomutase (PMM) is a key enzyme in prokaryotes and eukaryotes, which catalyzes the conversion of α-D-mannose 6-phosphate to α-D-mannose 1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for many metabolic pathways in the cells. We report here on the isolation of a gene from a genomic library of Sphingomonas chungbukensis DJ77, the pmmC gene encoding phosphomannomutase. The gene was cloned into E. coli expression vector, and the sequence was analyzed. The ribosomal binding site GGAAG lays 5 bp upstream of the ORF of 750 bp, which is initiated by ATG codon and terminated by TAG. The predicted sequence of the enzyme consists of 249 amino acids with a molecular mass of 27.4 kDa and showed 86.9% similarity to that of eukaryotic phosphomannomutase after bioinformatical analyses with the conserved domain search of NCBI. The purified gene product revealed the activity of phosphomannomutase. In conclusion, we confirmed that pmmC gene encodes phosphomannomutase actually.
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