Nagata Y, Imai R, Sakai A, Fukuda M, Yano K, Takagi M
Department of Agricultural Chemistry, University of Tokyo, Japan.
Biosci Biotechnol Biochem. 1993 May;57(5):703-9. doi: 10.1271/bbb.57.703.
Pseudomonas paucimobilis UT26 grows on gamma-hexachlorocyclohexane (gamma-HCH) as a sole source of carbon and energy. Tn5 mutation was introduced into UT26, and two kinds of mutants defective in gamma-HCH degradation were phenotypically isolated; one (UT64) completely lacked the activity to degrade gamma-HCH, while the other (UT61) retained a very low level of activity. Tagging and sequencing analysis showed that both mutants had a Tn5 insertion at the same site of the linA (gamma-HCH dehydrochlorinase encoding) gene. However, UT61 had an additional rearrangement, which could be the cause of its retaining a low level of activity. An in vitro complementation test with a crude extract from UT64 plus partially purified LinA protein showed that LinA was essential not only for the first-step reaction (gamma-HCH to gamma-pentachloro-cyclohexene; gamma-PCCH), but also for the second-step reaction (gamma-PCCH to compound B) of gamma-HCH degradation in UT26.
少动假单胞菌UT26能以γ-六氯环己烷（γ-HCH）作为唯一碳源和能源生长。将Tn5突变引入UT26，从表型上分离出两种γ-HCH降解缺陷型突变体；一种（UT64）完全丧失了降解γ-HCH的活性，而另一种（UT61）保留了极低水平的活性。标签和测序分析表明，两种突变体在linA（编码γ-HCH脱氯化氢酶）基因的同一位点都有一个Tn5插入。然而，UT61有一个额外的重排，这可能是其保留低水平活性的原因。用UT64的粗提物加部分纯化的LinA蛋白进行的体外互补试验表明，LinA不仅对UT26中γ-HCH降解的第一步反应（γ-HCH转化为γ-五氯环己烯；γ-PCCH）至关重要，而且对第二步反应（γ-PCCH转化为化合物B）也至关重要。
