Lang Gang-hua, Ogawa Naoto, Tanaka Yusuke, Fujii Takeshi, Fulthorpe Roberta R, Fukuda Masao, Miyashita Kiyotaka
National Institute for Agro-Environmental Sciences, 3-1-3 Kan-nondai, Tsukuba, Ibaraki 305-8604, Japan.
Biochem Biophys Res Commun. 2005 Jul 15;332(4):941-8. doi: 10.1016/j.bbrc.2005.05.045.
Two kinds of chlorocatechol 1,2-dioxygenase (CCD), TfdC and TfdC2 were detected in Sphingomonas sp. strain TFD44. These two CCDs could be simultaneously synthesized in TFD44 during its growth with 2,4-D as the sole carbon and energy sources. The apparent subunit molecular masses of TfdC and TfdC2 estimated by SDS-PAGE analysis were 33.8 and 33.1 kDa, respectively. The genes encoding the two CCDs were cloned and expressed in Escherichia coli. The two purified CCDs showed broad substrate specificities but had different specificity patterns. TfdC showed the highest specificity constant for 3-chlorocatechol and TfdC2 showed the highest specificity constant for 3,5-dichlorocatechol. The substrate specificity difference seemed to correlate with the alternation of amino acid supposed to be involved in the interaction with substrates. Whereas phylogenetic analysis indicated that the CCDs of Sphingomonas constitute a distinctive group among Gram-negative bacteria, TfdC and TfdC2 of TFD44 have divergently evolved in terms of their substrate specificity.
在鞘氨醇单胞菌属菌株TFD44中检测到了两种氯邻苯二酚1,2 -双加氧酶（CCD），即TfdC和TfdC2。在TFD44以2,4 -二氯苯氧乙酸作为唯一碳源和能源生长的过程中，这两种CCD能够同时合成。通过SDS - PAGE分析估计，TfdC和TfdC2的表观亚基分子量分别为33.8 kDa和33.1 kDa。编码这两种CCD的基因被克隆并在大肠杆菌中表达。两种纯化后的CCD均表现出广泛的底物特异性，但具有不同的特异性模式。TfdC对3 -氯邻苯二酚表现出最高的特异性常数，而TfdC2对3,5 -二氯邻苯二酚表现出最高的特异性常数。底物特异性差异似乎与推测参与底物相互作用的氨基酸变化有关。系统发育分析表明，鞘氨醇单胞菌的CCD在革兰氏阴性菌中构成一个独特的群体，而TFD44的TfdC和TfdC2在底物特异性方面已经发生了分歧进化。
