Bacillus megaterium으로부터 얻은 cytochrome P450 BM3 (CYP102A1)는 지방산 hydroxylase이며 monooxygenase enzymes의 매우 큰 superfamily이다. BM3에서 reductase domain은 fusion되어 있으며 heme domain을 촉매작용을 한다. 이 효소는 높은 촉매활성을 가지며 E.coli에서 쉽게 발현이 되고 높은 안정성도 보인다. ...
Bacillus megaterium으로부터 얻은 cytochrome P450 BM3 (CYP102A1)는 지방산 hydroxylase이며 monooxygenase enzymes의 매우 큰 superfamily이다. BM3에서 reductase domain은 fusion되어 있으며 heme domain을 촉매작용을 한다. 이 효소는 높은 촉매활성을 가지며 E.coli에서 쉽게 발현이 되고 높은 안정성도 보인다. P450 BM3는 lauric acid, myristic acid, palmitic acid를 (ω-1), (ω-2), and (ω-3) 위치에서 hydroxylation 시킨다. 이번 연구에서는 P450 BM3의 자연적 변형에 대해 알아보았다. 먼저, ATCC, KCTC 균주 기탁 기관으로부터 21종의 다른 bacillus megaterium을 얻어 PCR과 cloning 과정을 통해 BM3 variants를 찾았다. 그 중에서 17종이 PCR 되어 BM3와 유사한 결과물을 얻었고 17종 중 4종은 BM3 wild-type과 DNA sequence까지 완전히 일치하였다. 나머지 13종은 BM3 wild-type과 다른 변형을 보였다. 이런 variants는 전체 1050개 아미노산중 31개 아미노산이 바뀌었는데 이는 전체 아미노산 서열의 3%정도 되는 변형을 나타낸다. P450 BM3 variants의 아미노산 서열의 계통 발생적 분석을 통해 두 분류로 나뉜 것을 알 수 있는데 3개의 variants는 wild-type과 가깝고 7개의 variants는 다른 분류로 나뉘었다. CYP102A1.6, CYP102A1.8, CYP102A1.9, 그리고 CYP102A1.10 variants는 lauric acid를 hydroxylation시키는 regioselectivity pattern이 P450 BM3 wild type과 다르다. electron transfer되는 rate을 reductase domain을 통해 측정할 수 있는데 electron transfer rate이 variants가 P450 BM3WT보다 빠르다. 이를 통해 monooxygenation rate이 증가한다는 것을 설명할 수 있다. variants에서의 유전적 그리고 생화학적 정보는 다양한 기질에 대한 촉매활성을 갖는 engineering self-sufficient CYPs를 이해하는데 도움이 될 것으로 기대한다. 그리고 natural products의 bioconversion과 organic chemicals의 biodegradation에 유용할 것이다.
Bacillus megaterium으로부터 얻은 cytochrome P450 BM3 (CYP102A1)는 지방산 hydroxylase이며 monooxygenase enzymes의 매우 큰 superfamily이다. BM3에서 reductase domain은 fusion되어 있으며 heme domain을 촉매작용을 한다. 이 효소는 높은 촉매활성을 가지며 E.coli에서 쉽게 발현이 되고 높은 안정성도 보인다. P450 BM3는 lauric acid, myristic acid, palmitic acid를 (ω-1), (ω-2), and (ω-3) 위치에서 hydroxylation 시킨다. 이번 연구에서는 P450 BM3의 자연적 변형에 대해 알아보았다. 먼저, ATCC, KCTC 균주 기탁 기관으로부터 21종의 다른 bacillus megaterium을 얻어 PCR과 cloning 과정을 통해 BM3 variants를 찾았다. 그 중에서 17종이 PCR 되어 BM3와 유사한 결과물을 얻었고 17종 중 4종은 BM3 wild-type과 DNA sequence까지 완전히 일치하였다. 나머지 13종은 BM3 wild-type과 다른 변형을 보였다. 이런 variants는 전체 1050개 아미노산중 31개 아미노산이 바뀌었는데 이는 전체 아미노산 서열의 3%정도 되는 변형을 나타낸다. P450 BM3 variants의 아미노산 서열의 계통 발생적 분석을 통해 두 분류로 나뉜 것을 알 수 있는데 3개의 variants는 wild-type과 가깝고 7개의 variants는 다른 분류로 나뉘었다. CYP102A1.6, CYP102A1.8, CYP102A1.9, 그리고 CYP102A1.10 variants는 lauric acid를 hydroxylation시키는 regioselectivity pattern이 P450 BM3 wild type과 다르다. electron transfer되는 rate을 reductase domain을 통해 측정할 수 있는데 electron transfer rate이 variants가 P450 BM3WT보다 빠르다. 이를 통해 monooxygenation rate이 증가한다는 것을 설명할 수 있다. variants에서의 유전적 그리고 생화학적 정보는 다양한 기질에 대한 촉매활성을 갖는 engineering self-sufficient CYPs를 이해하는데 도움이 될 것으로 기대한다. 그리고 natural products의 bioconversion과 organic chemicals의 biodegradation에 유용할 것이다.
Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium, a fatty acid hydroxylase is a member of a very large superfamily of monooxygenase enzymes. In P450 BM3, the reductase domain is fused to its catalytic heme domain, so coexpression of a separate redox partner is not required. Furthermore, this ...
Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium, a fatty acid hydroxylase is a member of a very large superfamily of monooxygenase enzymes. In P450 BM3, the reductase domain is fused to its catalytic heme domain, so coexpression of a separate redox partner is not required. Furthermore, this enzyme has the highest catalytic activity ever recorded for P450-catalyzed reactions, is easily overexpressed in Escherichia coli, and has a high stability. P450 BM3 catalyses the hydroxylation of lauric acid, myristic acid, palmitic acid at the (ω-1), (ω-2), and (ω-3) positions. In this work, the possibility of the presence of natural variants of P450 BM3 has been examined. At first 21 different strains of B. megaterium obtained from ATCC and KCTC were screened if they have BM3 variants with typical PCR and cloning techniques. It was found that seventeen(17) strains have BM3-like PCR products. Their DNA sequence were determined, and 4 strains among 17 strains showed exactly matched DNA sequence with BM3WT. Thirteen(13) strains showed some variation from BM3WT. The amino acid changes of variants were from seventeen(17) to thirty one(31) among total 1,050 amino acids of BM3WT. These analyse indicate that the variation of natural BM3 enzymes can go up to 3% difference of amino acid sequence. Phylogenetic analysis of the amino acid sequences of P450 BM3 variants showed three variants are closely related and seven variants are distinct from BM3WT. Variants CYP102A1.6, CYP102A1.8, CYP102A1.9, and CYP102A1.10, the patterns of regioselectivity of hydroxylation of lauric acid are different from that of P450 BM3 wild-type (BM3WT). Measurement of the rate of electron transfer through the reductase domain reveals that it is significantly faster in the variants than in BM3WT, providing a likely explanation for the increased monooxygenation rate. The genetic and biochemical information on variants are expected to be helpful for engineering self-sufficient CYPs with broader catalytic activities towards various substrates, which would be useful for bioconversion of natural products and biodegradation of organic chemicals.
Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium, a fatty acid hydroxylase is a member of a very large superfamily of monooxygenase enzymes. In P450 BM3, the reductase domain is fused to its catalytic heme domain, so coexpression of a separate redox partner is not required. Furthermore, this enzyme has the highest catalytic activity ever recorded for P450-catalyzed reactions, is easily overexpressed in Escherichia coli, and has a high stability. P450 BM3 catalyses the hydroxylation of lauric acid, myristic acid, palmitic acid at the (ω-1), (ω-2), and (ω-3) positions. In this work, the possibility of the presence of natural variants of P450 BM3 has been examined. At first 21 different strains of B. megaterium obtained from ATCC and KCTC were screened if they have BM3 variants with typical PCR and cloning techniques. It was found that seventeen(17) strains have BM3-like PCR products. Their DNA sequence were determined, and 4 strains among 17 strains showed exactly matched DNA sequence with BM3WT. Thirteen(13) strains showed some variation from BM3WT. The amino acid changes of variants were from seventeen(17) to thirty one(31) among total 1,050 amino acids of BM3WT. These analyse indicate that the variation of natural BM3 enzymes can go up to 3% difference of amino acid sequence. Phylogenetic analysis of the amino acid sequences of P450 BM3 variants showed three variants are closely related and seven variants are distinct from BM3WT. Variants CYP102A1.6, CYP102A1.8, CYP102A1.9, and CYP102A1.10, the patterns of regioselectivity of hydroxylation of lauric acid are different from that of P450 BM3 wild-type (BM3WT). Measurement of the rate of electron transfer through the reductase domain reveals that it is significantly faster in the variants than in BM3WT, providing a likely explanation for the increased monooxygenation rate. The genetic and biochemical information on variants are expected to be helpful for engineering self-sufficient CYPs with broader catalytic activities towards various substrates, which would be useful for bioconversion of natural products and biodegradation of organic chemicals.
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